Methods and compositions for treating cancer

ABSTRACT

A nucleic acid sequence is provided that encodes a chimeric protein comprising a ligand that comprises a naturally occurring or modified follicle stimulating hormone sequence, e.g., an FSHβ sequence, or fragment thereof, which ligand binds to human follicle stimulating hormone (FSH) receptor, linked to either (a) a nucleic acid sequence that encodes an extracellular hinge domain, a transmembrane domain, a co-stimulatory signaling region, and a signaling endodomain; or (b) a nucleic acid sequence that encodes a ligand that binds to NKG2D. The vector containing the nucleic acid sequence, the chimeric proteins so encoded, and modified T cells expressing the chimeric protein, as well as method of using these compositions for the treatment of FSHR-expressing cancers or tumor cells are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 15/515,442,filed Mar. 29, 2017, which is a National Stage Entry under 35 U.S.C. 371of International Patent Application No. PCT/US2015/053128, filed Sep.30, 2015, which claims priority to U.S. Provisional Application No.62/059,068, filed Oct. 2, 2014 and U.S. Provisional Application No.62/202,824, filed Aug. 8, 2015. These applications are incorporated byreference herein.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“WST150PCT_ST25.txt”, was created on Sep. 28, 2015, and is 23 KB.

BACKGROUND OF THE INVENTION

Despite the advances in surgical approach and chemotherapy, the 5 yearsurvival of ovarian cancer has barely changed in the last 40 years.Immune pressure against ovarian cancer progression is elicited by tumorinfiltrating T cells. Despite the devastating course of ovarian cancer,T cells can spontaneously exert clinically relevant pressure againstmalignant progression, to the point that the pattern and the intensityof T cell infiltration can predict the patient's outcome. Ovariancancers are therefore immunogenic and optimal targets for the design ofnovel immunotherapies.

Over the last years, immunotherapy has emerged as a promising tool inthe treatment of cancer. For example, Chimeric Antigen Receptor (CAR)therapy has shown excellent results in the treatment of chemotherapyresistant hematologic malignancies. However, the paucity of specificantigens expressed on the surface of tumor cells that are not sharedwith healthy tissues, has so far prevented the success of thistechnology against most solid tumors, including ovarian cancer. There isconsiderable difficulty in finding specific antigens in tumor cellswhich are not present in normal tissues and elicit intolerable sideeffects. Additionally, the immunosuppressive effect of the tumormicroenvironment of solid tumors heavily impairs antitumor T cellresponses

SUMMARY OF THE INVENTION

Compositions and methods are described herein that provide effective anduseful tools and methods for the treatment of cancer, including solidtumors that are characterized by the cellular expression of theendocrine receptor, Follicle Stimulating Hormone (FSHR).

In one aspect, a nucleic acid construct comprises a nucleic acidsequence that encodes a chimeric protein comprising a ligand thatcomprises a follicle stimulating hormone (FSH) sequence, which ligandbinds to human FSHR, linked to sequences providing T cell activatingfunctions. In one aspect, the sequences providing T cell activatingfunctions are (a) a nucleic acid sequence that encodes an extracellularhinge domain, a spacer element, a transmembrane domain, a co-stimulatorysignaling region, and a signaling endodomain; or (b) a nucleic acidsequence that encodes an optional spacer and a ligand that binds toNKG2D. In one embodiment, the ligand is naturally occurring FSH, asingle subunit of FSH, FSHβ, an FSH or FSHβ fragment, or a modifiedversion of any of the foregoing sequences.

In another aspect, a chimeric protein comprising a ligand that comprisesan FSH sequence, which ligand binds to human FSHR, linked to either (a)an extracellular hinge domain, a transmembrane domain, a co-stimulatorysignaling region, and a signaling endodomain; or (b) an optional spacer,and a ligand that binds to NKG2D. In one embodiment, the ligand isnaturally occurring FSH, a single subunit of FSH, FSHβ, an FSH or FSHβfragment, or a modified version of any of the foregoing sequences.

In another aspect, a modified human T cell comprises a nucleic acidsequence that encodes a chimeric protein comprising a ligand thatcomprises an FSH sequence, which ligand binds to human FSHR, linked toan extracellular hinge domain, a transmembrane domain, a co-stimulatorysignaling region, and a signaling endodomain in a pharmaceuticallyacceptable carrier. In one embodiment, the modified T cell is anautologous T cell isolated from the patient to whom the T cell will bereadministered once the T cell is modified to contain a nucleic acidconstruct as described herein. In another embodiment, the modified Tcell is a universal allogeneic platform, i.e., a heterologous T cell,for administration to any number or patients once the T cell is modifiedas described herein.

In one embodiment, the ligand is naturally occurring FSH, a singlesubunit of FSH, FSHβ, an FSH or FSHβ fragment, or a modified version ofany of the foregoing sequences. In still another aspect, a method oftreating a cancer in a human subject comprises administering to thesubject in need thereof, a composition as described herein, includinge.g., a nucleic acid sequence, chimeric protein, or modified T cell. Inone embodiment, the method comprises administering to a subject in needthereof a modified human T cell that comprises a nucleic acid sequencethat encodes a chimeric protein comprising a ligand that comprises anFSH sequence, which ligand binds to human FSHR, an extracellular hingedomain, a transmembrane domain, a co-stimulatory signaling region, and asignaling endodomain.

In still another aspect, a method of treating a cancer in a humansubject comprises administering to a subject, a composition comprising anucleic acid sequence that encodes a chimeric protein comprising aligand that comprises an FSH sequence, which ligand binds to human FSHR,linked to either (a) a nucleic acid sequence that encodes anextracellular hinge domain, a spacer element, a transmembrane domain, aco-stimulatory signaling region, and a signaling endodomain; or (b) anucleic acid sequence that encodes an optional spacer and a ligand thatbinds to NKG2D. In one embodiment, the ligand is naturally occurringFSH, a single subunit of FSH, FSHβ, an FSH or FSHβ fragment, or amodified version of any of the foregoing sequences.

In still another aspect, a method of treating a cancer in a humansubject comprises administering to a subject, a composition comprising achimeric protein comprising a ligand that comprises an FSH sequence,which ligand binds to human FSHR, linked to either (a) a nucleic acidsequence that encodes an extracellular hinge domain, a spacer element, atransmembrane domain, a co-stimulatory signaling region, and a signalingendodomain; or (b) a nucleic acid sequence that encodes an optionalspacer and a ligand that binds to a tumor-associated NKG2D receptor.

In another aspect, a method of treating ovarian cancer comprisesadministering to a subject in need thereof, a modified human T cellcomprises a nucleic acid sequence that encodes a chimeric proteincomprising a ligand that comprises an FSH sequence, which ligand bindsto human FSHR, linked to an extracellular hinge domain, a transmembranedomain, a co-stimulatory signaling region, and a signaling endodomain ina pharmaceutically acceptable carrier. In one embodiment, the ligand isnaturally occurring FSH, a single subunit of FSH, FSHβ, an FSH or FSHβfragment, or a modified version of any of the foregoing sequences. Inanother embodiment, the female subject has been surgically treated forremoval of the ovaries prior to the administering step.

Other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of one construct (i.e., a nucleic acid constructor amino acid construct) that expresses the chimeric endocrine receptor(CER), FSH ligand protein, in T cells.

FIG. 2A is a bar graph showing that T cells containing the nucleic acidsequence encoding the chimeric protein expressing the ligand to FSHreceptor (FSHCER-CD8) respond specifically to FSHR-expressing B7F tumorcells. Positively transduced (GFP+) T cells carrying the FSHR-targetingCER (FSHCER-CD8; small checkerboard pattern) of FIG. 1 or an irrelevantmesothelin-targeting (K1) CAR (large checkerboard pattern) wereco-incubated (1:20) for 6 hours with ID8-DeJb29/Vegf-a tumor cells (B7)transduced with FSHCER-CD8 or an empty vector (ID-8; Irrelevant CAR).IFN-γ was quantified in supernatants (pg/mL). The FSH chimericprotein-transfected (or FSHR-targeting) modified T cells secretedinterferon-γ, an activation marker, in response to mouse B7 ovariantumor cells that overexpress FSHR. The mesothelin-targeting T cells donot secrete Interferon-γ against the FSHR-overexpressing tumor cell lineB7F. Likewise the T cell expressing the FSH chimeric protein is notactivated with a tumor cell B7 that does not express FSHR.

FIG. 2B is a bar graph showing the same analysis with a FSHCER-CD4construct.

FIG. 3 is a graph showing that T cells containing the nucleic acidsequence encoding the chimeric protein expressing the ligand to FSHreceptor delays progression of established FSHR+ tumor cells. A7C11syngeneic (B6) breast cancer cells that overexpress FSHR wereadministered into the flank of two groups of mice (5 mice/group). Fourdays after tumor cell administration, 10⁶ FSHR-targeting modified Tcells (▪) or mock (pBMN) transduced T cells (▴) were adoptivelytransferred intraperitoneally. The progression of the tumor growth inthe mice treated with the chimeric protein-carrying T cells is delayed.

FIG. 4A is a bar graph showing CD4/CD8 ratio in the splenic cells ofmice injected with the chimeric protein or the mock pBMN T cells inequal numbers on day 16 post-administration of the T cells carrying thechimeric protein or the mock protein.

FIG. 4B is a bar graph showing the cell count of adoptively transferredT cells of the mice treated as described as in FIG. 4A on day 16.

FIG. 4C is a bar graph showing the individual spleen CD4 counts of thesplenic cells from the mice of FIG. 4A on day 16.

FIG. 4D is a graph showing the CD8 cell counts of the splenic cells fromthe mice of FIG. 4A on day 16. As indicated in the flow cytometric data(data graphs not shown) of these spleen cells gated on CD8 and CD4markers (not shown), these figures also demonstrate that there is anincreased number of transfected cells, both CD4 and CD8, in the spleenof mice administered the chimeric protein bearing T cells, as comparedto the mock protein bearing T cells. FIGS. 4A-4D show that there is anincreased number of transferred T cells in the spleen of mice injectedwith the FSH chimeric protein—carrying T cells compared to those of miceinjected with the mock protein-carrying T cells. Also a higher CD4/CD8ratio is detected in the spleen cells into which the chimericprotein—carrying T cells were transferred. This ratio is found to be agood marker of response against cancer in this cell model of ovariancancer.

FIG. 5 is a nucleic acid sequence SEQ ID NO: 1 and an amino acidsequence SEQ ID NO: 2 for a construct comprising the following fusedcomponents of Table 1:

TABLE 1 Nucleic Acids of Amino Acids of Component SEQ ID NO: 1 SEQ IDNO: 2 Human FSH Beta Signal  1-54  1-18 Human FSH beta  55-387  19-129Spacer 388-432 130-144 Human FSH alpha 433-801 145-267 Hinge from HumanCD8 802-936 268-312 Transmembrane domain from  937-1008 313-336 HumanCD8 Human intracellular region 1009-1134 337-378 from 4-1BB Human CD3 ZDomain 1135-1473 379-490

FIG. 6A is a schematic of a chimeric FSH-Letal construct, whichdemonstrates how it binds to a tumor cell and a NK cell or a T cell(e.g., a CD8 T cell, a gamma T cell or an NK T cell).

FIG. 6B is a nucleic acid sequence SEQ ID NO: 3 and an amino acidsequence SEQ ID NO: 4 for a construct comprising the following fusedcomponents of Table 2:

TABLE 2 Nucleic Acids of Amino Acids of Component SEQ ID NO: 3 SEQ IDNO: 4 Human FSH Beta Signal 1-3 1 Human FSH beta  4-336  2-112 Spacer337-381 113-127 Human FSH alpha 382-750 128-250 Spacer 751-795 251-265Human extracellular NKG2D ligand  796-1365 266-454 (Letal)

FIG. 6C is a nucleic acid sequence SEQ ID NO: 5 and an amino acidsequence SEQ ID NO: 6 for a construct comprising the following fusedcomponents of Table 3. Construct has a MW of 45.87 kD, and employsnoncutting enzyme sites AscI, BamHI, BcgI, BclI, ClaI, HindIII, KpnI,MfeI, MluI, NcoI, NdeI, NotI, PacI, PmeI, PsiI, PvuI, SacII, SalI, SfiI,SgfI, SpeI, SphI, XbaI, and XhoI.

TABLE 3 Nucleic Acids of Amino Acids of Component SEQ ID NO: 5 SEQ IDNO: 6 Mouse FSH Beta Signal 1-3 1 Mouse FSH beta  4-336  2-112 Spacer337-381 113-127 Mouse FSH alpha 382-657 128-219 Spacer 658-708 220-236Extracellular domain of Mouse  709-1260 237-446 MULT1

FIG. 6D is a nucleic acid sequence SEQ ID NO: 7 and an amino acidsequence SEQ ID NO: 8 for a construct comprising the following fusedcomponents of Table 4:

TABLE 4 Nucleic Acids of Amino Acids of Component SEQ ID NO: 7 SEQ IDNO: 8 Human FSH Beta Signal 1-3 1 Human FSH beta  4-336  2-112 Spacer337-381 113-127 Human FSH alpha 382-657 128-219 Spacer 658-702 220-234Human extracellular domain of Letal  703-1272 235-423

FIG. 7A is a bar graph showing that adherent FSHR-transduced ID8-DeJb29Vegf-a (B7F) cancer cells were incubated for 24 hours with FSHCER-expressing or mocked transduced T cells, pBMN (1:40 ratio). Afterremoving non-adherent cells, trypan blue negative cells were counted ina hematocytometer. FSH-targeted CER T cells effectively eliminate FSHR⁺tumor cells.

FIG. 7B is a bar graph showing that adherent FSHR-transduced A7C11Ftransduced with the FSH-targeted constructs described herein effectivelyeliminate FSHR+ tumor cells. Adherent FSHR-transduced A7C11F cancercells were incubated for 24 hours with FSH CER-expressing or mockedtransduced T cells (1:40 ratio). After removing non-adherent cells,trypan blue negative cells were counted in a hematocytometer.FSH-targeted CER T cells effectively eliminate FSHR⁺ tumor cells.

FIG. 8 is a schematic showing variants of the FSH Chimeric EndocrineReceptor (CER)-T constructs described herein.

FIG. 9 is a graph showing the human FSHCER T cells kill ovarian tumorcells in a dose-dependent manner. HLA-A2+ human T cells were expandedwith ConA, spininfected with hFSHCER in pBMN with retronectin ormock-transduced at 20 and 44 hours, and kept at 0.5-1 million cells/mLwith 1 ug/mL of IL-7 and 20 U/mL of IL-2. At day 7, CER and control Tcells were sorted on GFP expression and rested for 18 hours, beforebeing plated with plated with HLA-A2+ human OVCAR-3 ovarian cancer cells(10000 per well; spontaneously FSHR+) on the indicated effector (E) totarget (T) ratios. Six hours after setting the coculture cells werestained with Annexin V and 7AAD and cytotoxicity was analyzed by flowcytometry. The percentage of specific lysis was calculated as(experimental dead−spontaneous dead)/(maximum dead−spontaneousdead)×100%.

FIG. 10 is a Western gel showing that advanced human ovarian carcinomaspecimens express variable levels of FSHR. FSHR protein expression wasanalyzed by Western Blot (Santa Cruz #H-190) in 6 unselected humanadvanced ovarian carcinoma specimens, and compared to that inFSH-targeted CER T cell-sensitive OVCAR3 cells. β-actin Ab, Sigma #A5441

FIG. 11 is a graph showing that FSHCER T cells abrogate the progressionof fshr-expressing orthotopic ovarian tumors. T cells carryingFSHR-targeting cars (FSH-CER) or identically expanded mock-transduced tcells (PBMN) were intraperitoneally administered at days 7 and 14 afterintraperitoneal challenge with ID8-deJb29/vegf-a tumor cells transducedwith FSHR (n=5 mice/group). Malignant progression was compared.

FIG. 12 is a graph showing that a modified allogeneic or heterologoushuman FSH CER T cell generated by using TALL-103/2 cells, kill ovariantumor cells in a dose-dependent manner. TALL-103/2 cells were transducedwith hFSHCER in pBMN and maintained in culture with 20 U/mL of IL-2. FSHCER-transduced (▪) or mock-transduced (▴) TALL-103/2 cells were deprivedfrom IL-2 24 h before being incubated with luciferase-transduced FSHR+human OVCAR-3 ovarian cancer cells (10000 per well) at the indicatedeffector (E) to target (T) ratios. Four hours after setting theco-culture cells were lysed and luciferase signal quantified. Thepercentage of specific lysis was calculated as (experimentaldead−spontaneous dead)/(maximum dead−spontaneous dead)×100%.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods are provided herein that elicit protectiveanti-tumor immunity against, and prevent recurrence of, e.g., ovariancancer or other cancers characterized by tumor cells bearing the FSHreceptor (FSHR), e.g., prostate cancer cells⁵² and metastatic tumorlesions⁵¹. By targeting hormone receptors by taking advantage ofendogenous ligands as targeting motifs, challenges that have preventedthe success of certain immunotherapy technologies against epithelialtumors are overcome.

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The following definitions are provided for clarityonly and are not intended to limit the claimed invention.

Follicle stimulating hormone (FSH) is a central hormone of mammalianreproduction produced primarily in the anterior pituitary gland. Thishormone exerts its normal biological role by binding to the plasmamembrane follicle-stimulating hormone receptor (FSHR) and stimulatingfollicular maturation and estrogen production in females. In males, theinteraction of FSH and FSHR stimulates Sertoli cell proliferation andmaintenance of normal spermatogenesis. The naturally occurring FSHhormone is a heterodimer formed of two subunits, an alpha and a betasubunit. The alpha subunit is also referred to as CGα, and is common toluteinizing hormone (LH) and thyroid stimulating hormone (TSH). Thenucleic acid and amino acid sequences of the alpha and beta subunits ofFSH for humans and other mammalian species are publically known andaccessible.

FSHR is a hormone receptor that is selectively expressed in women in theovarian granulosa cells and at low levels in the ovarian endothelium.Most importantly, this surface receptor is expressed in 50-70% ofovarian carcinomas but not in the brain, as negative feedback depends onsensing estrogen. Given that oophorectomy is a standard procedure in thetreatment of ovarian cancer, targeting the FSHR should not cause damageto healthy tissues.

As used herein, the phrase “a ligand that comprises an FSH sequence,which ligand binds FSHR” includes the naturally occurring full-lengthFSH sequence of a suitable mammal. The ligand comprises a sufficient FSHsequence to permit binding between the ligand and the FSHR via thenaturally affinity between the hormone sequence and the receptor. Theligand is not an antibody or antibody fragment and does not bind to thereceptor in that manner. If the ligand is naturally occurring, e.g., afull-length FSHβ-FSHα sequence or naturally occurring fragment thereof,the ligand does not induce an immunogenic reaction in the subject towhich it is administered. If the ligand comprises a modified full-lengthor fragment of the naturally occurring FSH sequence, in certainembodiments the modifications are not sufficient to induce any strongimmunogenic reaction within the subject to which the ligand isadministered.

In one embodiment, a ligand that comprises an FSH sequence is anaturally occurring full length human FSH, e.g., the FSHβ sequencelinked to the FSHα sequence. In another embodiment, a ligand thatcomprises an FSH sequence is a modified FSHβ sequence linked to anaturally occurring FSHα sequence. In another embodiment, a ligand thatcomprises an FSH sequence is a modified FSHβ sequence linked to amodified FSHα/CGα sequence. In another embodiment, the ligand is anaturally occurring FSHβ sequence linked to a modified FSHα sequence. Inanother embodiment, where the subject mammal is a human and the targettumor is a human tumor, a suitable FSH sequence is human FSH or modifiedversions of the human sequence. Alternatively the ligand is a modifiedFSH, such as a naturally occurring or modified FSHβ sequence linked viaan optional spacer to a naturally occurring or modified FSHα sequence.In another embodiment, the ligand is a single naturally occurring ormodified FSHβ subunit alone. In another embodiment, the ligand is anaturally occurring FSHβ subunit linked via an optional spacer sequenceto a modified second FSHβ sequence. In another embodiment, the ligand isa modified FSHβ subunit linked via an optional spacer sequence to anaturally occurring second FSHβ sequence.

In yet another embodiment, the ligand comprises a fragment of anaturally occurring or modified FSH sequence. In yet another embodiment,the ligand comprises a fragment of a naturally occurring or modifiedFSHβ sequence. In another embodiment, the ligand is a naturallyoccurring FSHβ subunit linked to a modified FSHα subunit or fragmentthereof. In another embodiment, the ligand is a modified FSHβ subunit orfragment thereof linked to a naturally occurring or FSHα subunit. Inanother embodiment, the ligand comprises a fragment of a naturallyoccurring or modified FSHβ sequence linked together.

By “naturally occurring” is meant that the sequence is a native nucleicacid or amino acid sequence that occurs in the selected mammal,including any naturally occurring variants in various nucleic acidand/or amino acid positions in the sequences that occur among variousmembers of the mammalian species.

By “modified” is meant that the reference sequence, e.g., FSH or afragment thereof or FSHβ linked to FSHα nucleic acid or amino acidsequence, or either subunit sequence individually has been deliberatelymanipulated. Suitable modifications include the use of fragments of thesequences shorter than the naturally occurring full length hormone. Suchmodifications include changes in the nucleic acid sequences to includepreferred codons, which may encode the same or a related amino acid thanthat occurring in the native amino acid sequence. Modifications alsoinclude changes in the nucleic acid or amino acid sequences to introduceconservative amino acid changes, e.g., a change from one charged orneutral amino acid for a differently charged amino acid. Suchmodifications may also include use of the FSHβ with or without FSHαsequence in a deliberately created fusion with other sequences withwhich FSHβ or FSHα do not naturally occur. Modifications also includelinking the subunits using deliberately inserted spacer sequences orlinking fragments of the subunits together or linking repetitivefragments or subunits together in fusions which are not naturallyoccurring.

As one example, a naturally occurring human FSHβ nucleic acid sequenceinclusive of the signal sequence comprises or consists of nucleic acids1-387 of SEQ ID NO: 1, and amino acid sequence is aa 1-129 of SEQ ID NO:2. The FSHβ signal sequence itself comprises or consists of nucleicacids 1-54 of SEQ ID NO: 1, and amino acid sequence is aa 1-18 of SEQ IDNO: 2 The mature FSHβ comprises or consists of nucleic acids 55-387 ofSEQ ID NO: 1, and amino acid sequence is aa 19-129 of SEQ ID NO: 2.

As another example for use in the methods and compositions herein, amature human FSHβ nucleic acid sequence comprises or consists of nucleicacids 4-336 of SEQ ID NO: 3 or 7, and amino acid sequence is aa 2-112 ofSEQ ID NO: 4 or 8. As another example for use in the methods andcompositions herein, a useful fragment of a human FSHβ nucleic acidsequence comprises or consists of nucleic acids 55-99 of FSHβ SEQ ID NO:1, nucleic acids 153-213 of FSHβ SEQ ID NO: 1, nucleic acids 207-249 ofFSHβ SEQ ID NO: 1, or nucleic acids 295-339 of FSHβ SEQ ID NO: 1. Asanother example for use in the methods and compositions herein, a usefulfragment of a human FSHβ amino acid sequence comprises or consists ofamino acids 19-33 of FSHβ SEQ ID NO: 2, amino acids 51-71 of FSHβ SEQ IDNO: 2, amino acids 69-83 of FSHβ SEQ ID NO: 2, or amino acids 99-113 ofFSHβ SEQ ID NO: 2.

In embodiments in which the ligand also comprises an FSHα sequence, thenaturally occurring human FSHα nucleic acid sequence comprises orconsists of nucleic acids 433-801 of SEQ ID NO: 1, and amino acidsequence is aa 145-267 of SEQ ID NO: 2. In another embodiment for use inthe methods and compositions herein, a human FSHα nucleic acid sequencecomprises or consists of nucleic acids 382-750 of SEQ ID NO: 3, andamino acid sequence is aa 128-250 of SEQ ID NO: 4. In another embodimentfor use in the methods and compositions herein, a fragment of a humanFSHα nucleic acid sequence comprises or consists of nucleic acids382-657 of SEQ ID NO: 7, and amino acid sequence is aa 128-219 of SEQ IDNO: 8.

It should be understood that amino acid modifications or nucleic acidmodifications as described above applied to these fragments are alsouseful ligands in this method. The ligand does not bind to FSHR in anantibody or antibody fragment—antigen complex. As described above, theligands described herein bind using the naturally affinity between thenatural hormone (or a modified version of a natural hormone) and itsreceptor. Because the ligand is a natural hormone or a modified versionthereof, it is designed to avoid inducing an antigenic response in thesubject.

The terms “linker” and “spacer” are used interchangeably and refer to anucleic acid sequence that encodes a peptide of sufficient length toseparate two components and/or refers to the peptide itself. Thecomposition and length of a linker may be selected depending upon theuse to which the linker is put. In one embodiment, an amino acid linkerused to separate the FSHα and FSHβ (either naturally occurring sequencesor modified sequences or fragments) is between 2 to 70 amino acids inlength, including any number within that range. For example, in oneembodiment the linker is 10 amino acids in length. In anotherembodiment, the linker is 15 amino acids in length. In still otherembodiment, the linker is 25, 35, 50 or 60 amino acids in length. See,for example, the spacers/linkers identified in the sequences describedin Tables 1-4 above.

Correspondingly, the nucleic acid sequences encoding the linker orspacer are comprised of from 6 to 210 nucleotides in length, includingall values in that range. In certain embodiment, the linker comprisesmultiple glycine residues or nucleic acids encoding them. In certainembodiments, the amino acid linker comprises multiple serine residues ornucleic acids encoding them. In other embodiment, the linker comprisesmultiple thymine residues or nucleic acids encoding them. In still otherembodiment, linkers and spacers comprise any combination of the serine,thymine and glycine residues. Still other linkers can be readilydesigned for use.

As used herein, a “vector” comprises any genetic element including,without limitation, naked DNA, a phage, transposon, cosmid, episome,plasmid, bacteria, or a virus, which expresses, or causes to beexpressed, a desired nucleic acid construct.

As used herein, the term “subject” or “patient” refers to a male orfemale mammal, preferably a human. However, the mammalian subject canalso be a veterinary or farm animal, a domestic animal or pet, andanimals normally used for clinical research. In one embodiment, thesubject of these methods and compositions is a human.

The term “cancer” as used herein means any disease, condition, trait,genotype or phenotype characterized by unregulated cell growth orreplication as is known in the art. A “cancer cell” is cell that dividesand reproduces abnormally with uncontrolled growth. This cell can breakaway from the site of its origin (e.g., a tumor) and travel to otherparts of the body and set up another site (e.g., another tumor), in aprocess referred to as metastasis. A “tumor” is an abnormal mass oftissue that results from excessive cell division that is uncontrolledand progressive, and is also referred to as a neoplasm. Tumors can beeither benign (not cancerous) or malignant. The compositions and methodsdescribed herein are useful for treatment of cancer and tumor cells,i.e., both malignant and benign tumors, so long as the cells to betreated express FSHR. Thus, in various embodiments of the methods andcompositions described herein, the cancer can include, withoutlimitation, breast cancer, lung cancer, prostate cancer, colorectalcancer, esophageal cancer, stomach cancer, bladder cancer, pancreaticcancer, kidney cancer, cervical cancer, liver cancer, ovarian cancer,and testicular cancer.

As used herein the term “pharmaceutically acceptable carrier” or“diluent” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, adjuvants and the like, compatible with administrationto humans. In one embodiment, the diluent is saline or buffered saline.The term “a” or “an”, refers to one or more, for example, “an anti-tumorT cell” is understood to represent one or more anti-tumor T cells. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” is usedinterchangeably herein. The term “about” is used herein to modify areference value and to include all values ±0.01% of that value up tovalues of ±10% of the reference value, and all numbers within andincluding these endpoints, e.g., ±0.5%, ±1%, ±5%, etc. Variousembodiments in the specification are presented using “comprising”language, which is inclusive of other components or method steps. When“comprising” is used, it is to be understood that related embodimentsinclude descriptions using the “consisting of” terminology, whichexcludes other components or method steps, and “consisting essentiallyof” terminology, which excludes any components or method steps thatsubstantially change the nature of the embodiment or invention.

In one embodiment, this invention provides a nucleic acid sequence thatencodes a chimeric protein comprising a ligand comprising an FSHsequence that binds to human FSHR, linked to nucleic acid sequences thatencode T cell activating functions. As described above in more detail,in certain embodiments, the ligand is a naturally occurring FSH withboth subunits, a single subunit of FSH, an FSHβ subunit only, anFSHα/CGα or FSHβ fragment, or a modified version of the foregoingsequences.

In one embodiment, the T cell activating functions can be provided bylinking the above noted ligand with nucleic acid sequences encodingcomponents useful in the design of known Chimeric Antigen Receptors(CAR). See, e.g., Sadelain, M et al, “The basic principles of chimericantigen receptor (CAR) design” 2013 April, Cancer Discov. 3(4): 388-398;International Patent Application Publication WO2013/044255, US patentapplication publication No. US 2013/0287748, and other publicationsdirected to the use of such chimeric proteins. These publications areincorporated by reference to provide information concerning variouscomponents useful in the design of some of the constructs describedherein. Such CAR T cells are genetically modified lymphocytes expressinga ligand that allows them to recognize an antigen of choice. Uponantigen recognition, these modified T cells are activated via signalingdomains converting these T cells into potent cell killers. An advantageover endogenous T cells is that they are not MHC restricted, whichallows these T cells to overcome an immune surveillance evasion tacticused in many tumor cells by reducing MHC expression¹⁹.

For example, such T cell activating functions can be provided by linkingthe ligand via optional spacers to transmembrane domains, co-stimulatorysignaling regions, and/or signaling endodomains.

Thus, one embodiment of a nucleic acid sequence useful in the methodsdescribed herein is exemplified in FIG. 5 SEQ ID NO: 1 and Table 1herein. The nucleic acid sequence or CER construct comprises a ligandformed of a naturally occurring human FSHβ sequence formed of the 18amino acid human FSHβ signal sequence and the 120 amino acid matureFSHβ, linked to a 15 amino acid spacer, and to the naturally occurring123 amino acid FSHα sequence. The CER construct also includes othercomponents, i.e., an extracellular hinge domain, a transmembrane domain,a human intracellular region and a signaling endodomain. In the case ofthe construct of FIG. 5, e.g. the hinge region and transmembrane domainsare from human CD8α, the human intracellular region is from 4-1BB, andthe signaling domain is the human CD3 ζ domain.

Other embodiments useful as such a nucleic acid construct can includethat construct with a different ligand, such as one of the ligandsdescribed above. In one embodiment, the FSHα sequence in the sameconstruct described in FIG. 5 may be a shortened sequence having thenucleic acid sequence of nts 382-657 of SEQ ID NO: 7, and amino acidsequence of aa 128-219 of SEQ ID NO: 8. Still another embodiment of theligand used in the construct of FIG. 5 may comprise the FSHβ sequencewithout signal sequence amino acids 2-18 of SEQ ID NO: 2. Embodimentssimilar to that of the nucleic acid construct of FIG. 5 may be readilydesigned by substituting the ligand portions of Table 1 with any of theligands, modified, naturally occurring or fragments discussed above.

Other embodiments of a nucleic acid construct similar to that of FIG. 5may employ different components, such as those detailed in Sadelain etal, cited above, or the patent publications, incorporated by referenceherein. For example, where a hinge domain is employed, other naturallyoccurring or synthetic hinge domains, including an immunoglobulin hingeregion, such as that from IgG1, the CH₂CH₃ region of immunoglobulin,fragments of CD3, etc. Other embodiments of a nucleic acid constructsimilar to that of FIG. 5 may employ a different naturally occurring orsynthetic transmembrane domain obtained from a T cell receptor. Varioustransmembrane proteins contain domains useful in the constructsdescribed herein. For example, transmembrane domains obtained fromT-cell receptors, CD28, CD3 epsilon, CD45, CD4, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 have been noted tobe useful.

Other embodiments of a nucleic acid construct similar to that of FIG. 5may employ a different naturally occurring or synthetic intracellularregion, including, among others known in the art, a costimulatorysignaling region. The costimulatory signaling region may be theintracellular domain of a cell surface molecule (e.g., a costimulatorymolecule) such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3. See, e.g., others listed in the publications cited above.

Other embodiments of a nucleic acid construct similar to that of FIG. 5may employ a different naturally occurring or synthetic cytoplasmicsignaling domain including, among others known in the art, those derivedfrom CD3 ζ, TCR ζ, FcR γ, FcR β, CD3γ, CD3 δ, CD3 ε, CD5, CD22, 25CD79a, CD79b, and CD66d, among others.

Given the teachings provided herein and using the information known tothe art, any number of variations of the nucleic acid constructs, suchas FIG. 5 may be designed for use in the methods described herein.

Thus, another component described herein is a chimeric proteincomprising a ligand that comprises an FSHβ sequence, or a modificationor fragment of said FSH sequence, which ligand binds to human FSHR,linked to peptides or proteins that have T cell activating functions.Such a chimeric protein comprises a ligand as described above that bindsto human FSHR linked to an extracellular hinge domain, a transmembranedomain, a co-stimulatory signaling region, and a signaling endodomain.Exemplary chimeric proteins are encoded by the nucleic acid sequencesdescribed above. One embodiment of such a chimeric protein is that ofFIG. 5 SEQ ID NO: 2. Others are readily designed employing the variousligands identified herein, e.g., one or more of the FSHβ fragmentidentified in detail above, or the other FSHR binding ligands identifiedherein in place of the ligand specific exemplified in SEQ ID NO: 2.

In another embodiment, a useful CER construct is a nucleic acid sequencethat encodes ligand that comprises an FSHβ sequence, or a modificationor fragment of said FSH sequence, which ligand binds to human FSHR, asdescribed above, linked to a nucleic acid sequence that encodes a ligandthat binds to a tumor-associated NKG2D receptor. See, e.g., FIG. 6A. Onesuch NKG2D ligand is termed Letal or ULBP4. Letal is encoded by nucleicacid sequence nts 796-1365 of SEQ ID NO: 3 and has the amino acidsequence of aa 266-454 of SEQ ID NO: 4. See, e.g., Conejo-Garcia, J etal, “Letal, A Tumor-Associated NKG2D Immunoreceptor Ligand, InducesActivation and Expansion of Effector Immune Cells” July 2003, Canc.Biol. & Ther., 2(4): 446-451; and US patent application publication No.20060247420, incorporated by reference herein. Other NKG2D ligands oramino acid modifications, modifications on the nucleic acid level orfunctional fragments of the Letal sequence may be substituted in thisdescription for the exemplified Letal sequences.

Additionally, these FSHR binding ligands and NKG2D ligand are optionallylinked by a suitable spacer or linker as described above.

Specific examples of such a nucleic construct are provided in FIG. 6A,FIG. 6B, Table 2, SEQ ID NO: 3, FIG. 6D, Table 4, SEQ ID NO: 7, and FIG.8. In the embodiment of FIG. 7B, the FSHR binding ligand is formed of anaturally occurring human FSHβ sequence formed of a single amino acidmethionine from the signal sequence, followed by the 120 amino acidmature FSHβ, linked to a 15 amino acid spacer, in turn linked to thenaturally occurring 123 amino acid FSHα sequence. This ligand is in turnlinked to Letal via another 15 amino acid spacer. In the embodiment ofFIG. 6D, the FSHR binding ligand is formed of a naturally occurringhuman FSHβ sequence formed of a single amino acid methionine from thesignal sequence, followed by the 120 amino acid mature FSHβ, linked to a15 amino acid spacer, in turn linked to the modified FSHα sequence,i.e., a fragment of amino acids 128-219 of SEQ ID NO: 8, encoded bynucleotides 382-657 of SEQ ID NO: 7. This ligand is in turn linked toLetal via another 15 amino acid spacer.

Other embodiments useful as such a nucleic acid construct can includethe constructs of FIGS. 6B and 6D with a different ligand-encodingsequence, such as a sequence encoding one of the ligands describedabove. In one embodiment, the FSHα sequence in the same constructdescribed in FIG. 6B may be a single or multiple copies of full lengthFSHβ with or without a signal sequence. As another example the constructof FIG. 6B or 6D may contain a ligand formed by a fragment of a humanFSHβ encoded by nucleic acid sequence comprising or consisting ofnucleic acids 55-99 of FSHβ SEQ ID NO: 1, nucleic acids 153-213 of FSHβSEQ ID NO: 1, nucleic acids 207-249 of FSHβ SEQ ID NO: 1, or nucleicacids 295-339 of FSHβ SEQ ID NO: 1. The ligand may be formed by thesefragments alone, in combination or substituted for the full-length FSHβand thus fused via a linker with the FSHα sequence of FIG. 6B or 6D.Embodiments similar to that of the nucleic acid construct of FIG. 6B or6D may be readily designed by substituting the ligand portions of Table2 or 4 with any of the ligands, modified, naturally occurring orfragments discussed above.

As another aspect, therefore, is a chimeric or bi-specific proteinencoded by the nucleic acid sequences described above and comprising aligand comprising a FSHβ sequence, or a modification or fragment of saidFSH sequence as described herein that binds to human FSHR, linked to aligand that binds to NKG2D. These proteins are primarily useful in theform of a protein, and function in vivo to bring together endogenouslymphocytes and FSHR⁺ tumor cells.

In still other aspects, recombinant vectors carrying the above-describednucleic acid constructs are provided. The nucleic acid constructs may becarried, and chimeric proteins may be expressed in, plasmid basedsystems, of which many are commercially available or in replicating ornon-replicating recombinant viral vectors. The nucleic acid sequencesdiscussed herein may be expressed and produced using such vectors invitro in desired host cells or in vivo. Thus, in one embodiment, thevector is a non-pathogenic virus. In another embodiment, the vector is anon-replicating virus. In one embodiment, a desirable viral vector maybe a retroviral vector, such as a lentiviral vector. In anotherembodiment, a desirable vector is an adenoviral vector. In still anotherembodiment, a suitable vector is an adeno-associated viral vector.Adeno, adeno-associated and lentiviruses are generally preferred becausethey infect actively dividing as well as resting and differentiatedcells such as the stem cells, macrophages and neurons. A variety ofadenovirus, lentivirus and AAV strains are available from the AmericanType Culture Collection, Manassas, Va., or available by request from avariety of commercial and institutional sources. Further, the sequencesof many such strains are available from a variety of databasesincluding, e.g., PubMed and GenBank.

In one embodiment, a lentiviral vector is used. Among useful vectors arethe equine infectious anemia virus and feline as well as bovineimmunodeficiency virus, and HIV-based vectors. A variety of usefullentivirus vectors, as well as the methods and manipulations forgenerating such vectors for use in transducing cells and expressingheterologous genes, e.g., N Manjunath et al, 2009 Adv Drug Deliv Rev.,61(9): 732-745; Porter et al., N Engl J Med. 2011 Aug. 25;365(8):725-33), among others.

In another embodiment, the vector used herein is an adenovirus vector.Such vectors can be constructed using adenovirus DNA of one or more ofany of the known adenovirus serotypes. See, e.g., T. Shenk et al.,Adenoviridae: The Viruses and their Replication”, Ch. 67, in FIELD'SVIROLOGY, 6^(th) Ed., edited by B. N Fields et al, (Lippincott RavenPublishers, Philadelphia, 1996), p. 111-2112; U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses; U.S. Pat. No.7,247,472; WO 2005/1071093, etc. One of skill in the art can readilyconstruct a suitable adenovirus vector to carry and express a nucleotideconstruct as described herein. In another embodiment, the vector usedherein is an adeno-associated virus (AAV) vector. Such vectors can beconstructed using AAV DNA of one or more of the known AAV serotypes.See, e.g., U.S. Pat. Nos. 7,803,611; 7,696,179, among others.

In yet another embodiment, the vector used herein is a bacterial vector.In one embodiment, the bacterial vector is Listeria monocytogenes. See,e.g., Lauer et al, Infect. Immunity, 76(8):3742-53 (August 2008). Thus,in one embodiment, the bacterial vector is live-attenuated orphotochemically inactivated. The chimeric protein can be expressedrecombinantly by the bacteria, e.g., via a plasmid introduced into thebacteria, or integrated into the bacterial genome, i.e., via homologousrecombination.

These vectors also include conventional control elements that permittranscription, translation and/or expression of the nucleic acidconstructs in a cell transfected with the plasmid vector or infectedwith the viral vector. A great number of expression control sequences,including promoters which are native, constitutive, inducible and/ortissue-specific, are known in the art and may be utilized. In oneembodiment, the promoter is selected based on the chosen vector. Inanother embodiment, when the vector is lentivirus, the promoter is U6,H1, CMV IE gene, EF-1α, ubiquitin C, or phosphoglycero-kinase (PGK)promoter. In another embodiment, when the vector is an AAV, the promoteris an RSV, U6, or CMV promoter. In another embodiment, when the vectoris an adenovirus, the promoter is RSV, U6, CMV, or H1 promoters. Inanother embodiment, when the vector is Listeria monocytogenes, thepromoter is a hly or actA promoter. Still other conventional expressioncontrol sequences include selectable markers or reporter genes, whichmay include sequences encoding geneticin, hygromicin, ampicillin orpurimycin resistance, among others. Other components of the vector mayinclude an origin of replication. Selection of these and other promotersand vector elements are conventional and many such sequences areavailable (see, e.g., the references cited herein).

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts (Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.),use of overlapping oligonucleotide sequences, polymerase chain reaction,and any suitable method which provides the desired nucleotide sequence.

Thus, in one embodiment, using the information taught herein andpublically available and known vector construction components andtechniques, one of skill in the art can construct a viral vector (orplasmid) that expresses the desired nucleic acid construct. The chimericproteins encoded by these nucleic acid constructs may be expressed invitro, or ex vivo in host cells or expressed in vivo by administrationto a mammalian subject. Alternatively the chimeric proteins may begenerated synthetically by known chemical synthesis methodologies. Oneof skill in the art can select the appropriate method to produce thesechimeric proteins depending upon the components, the efficiency of themethodologies and the intended use, e.g., whether for administration asproteins, nucleic acids or in adoptive T cells, or otherwise toaccomplish the desired therapeutic result.

In yet another aspect, a modified human T cell is provided thatcomprises a nucleic acid sequence that encodes a chimeric proteincomprising a ligand that binds to human FSHR, linked to nucleic acidsequences that encode T cell activating functions. In one embodiment,these latter nucleic acid sequences encode an extracellular hingedomain, a transmembrane domain, a co-stimulatory signaling region, and asignaling endodomain in a pharmaceutically acceptable carrier.

A modified T cell is a T cell that has been transduced or transfectedwith one of the above-described vectors carrying the nucleic acidconstructs encoding the chimeric proteins. Desirably, the T cell is aprimary T cell, a CD8 (cytotoxic) T cell, or an NK T cell or other Tcell obtained from the same mammalian subject into whom the modified Tcell is administered or from another member of the mammalian species. Inone embodiment, the T cell is an autologous human T cell or naturalkiller (NK) T cell obtained from the subject or from a bone marrowtransplant match for the subject. Other suitable T cells include T cellsobtained from resected tumors, a polyclonal or monoclonal tumor-reactiveT cell. The T cell is generally obtained by apheresis, and transfectedor transduced with the selected nucleic acid construct to express thechimeric protein in vivo.

Still other suitable T cells include an allogeneic or heterologous Tcells useful as a universal T cell platform carrying the nucleic acidconstructs described herein. In one embodiment, a human cytotoxic T cellmay be employed. TALL-104 and TALL-103/2 cells are CD3-responsivelymphocytes, CD3⁺TCRαβ⁺ and CD3⁺TCRγδ⁺, respectively, derived fromchildhood T cell leukemia that display major histocompatibility complexnonrestricted, NK cell receptor-mediated tumoricidal activity, primarilydependent on NKG2D.^(59, 60, 61) TALL cells display a broad range oftumor target reactivity that is NKG2D-dependent. Irradiated TALL-104cells have been used for the treatment of metastatic breast and ovariancancer due to their spontaneous (NK-like) cytolytic activity and safety.

These modified T cells, whether autologous or endogenous, are activatedvia signaling domains converting these T cells into potent cell killers.The autologous cells have an advantage over endogenous T cells in thatthey are not MHC restricted, which allows these T cells to overcome animmune surveillance evasion tactic used in many tumor cells by reducingMHC expression. The endogenous cells, such as the TALL cells, have anadvantage in being universal, amenable to mass production,standardization and further cell engineering, to target FSHR⁺ ovariancancers.

In yet another embodiment, the modified T cell is also engineered exvivo to inhibit, ablate, or decrease the expression of Forkhead BoxProtein (Foxp1). In one embodiment, The T cells is engineered ormanipulated to decrease Foxp1 before the T cell is transfected with anucleic acid sequence as described above that encodes the chimericprotein comprising a ligand that comprises a naturally occurring ormodified FSH sequence or fragment thereof, which ligand binds to humanFSHR, linked to other T cell stimulating or targeting sequences. Inanother embodiment the manipulation to decrease or ablate Foxp1 occursafter the T cell is transfected with a nucleic acid sequence thatencodes a chimeric protein or bi-specific protein as described herein.In one embodiment, the T cell has been pre-treated so that it does notexpress Foxp1 once the T cells are delivered to the subject. Mostdesirably, the Foxp1 in the modified T cell is ablated. The T cells maybe treated with zinc finger nucleases, transcription activator-likeeffector nucleases (TALEN), the CRISPR/Cas system, or engineeredmeganuclease re-engineered homing endonucleases along with sequencesthat are optimized and designed to target the unique sequence of Foxp1to introduce defects into or delete the Fox-P1 genomic sequence. Bytaking advantage of endogenous DNA repair machinery, these reagentsremove Foxp1 from the modified T cells before adoptive transfer.Alternatively, the T cells may be co-transfected with another nucleicacid sequence designed to inhibit, decrease, down-regulate or ablateexpression of Foxp1. See, e.g., International Patent ApplicationPublication WO2013/063019, incorporated by reference herein. Variouscombinations of these techniques may also be employed before or afterthe T cells have been modified by introduction of the nucleic acidconstruct.

Generally, when delivering the vector by transfection to the T cells,the vector is delivered in an amount from about 5 μg to about 100 μg DNAto about 1×10⁴ cells to about 1×10¹³ cells. In another embodiment, thevector is delivered in an amount from about 10 to about 50 μg DNA to1×10⁴ cells to about 1×10¹³ cells. In another embodiment, the vector isdelivered in an amount from about 5 μg to about 100 μg DNA to about 10⁵cells. However, the relative amounts of vector DNA to the T cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected. The vector may beintroduced into the T cells by any means known in the art or asdisclosed above, including transfection, transformation, infection,extraporation or direct DNA injection. The nucleic acid construct may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently.

The resulting modified T cells are prepared to expressed the nucleicacid constructs for adoptive therapy in a suitable pharmaceuticalcarrier. However, the chimeric bi-specific proteins may be administeredas proteins in a suitable pharmaceutical carrier, as mentioned above.

All of the compositions and components described above may be used inthe methods described herein for treating the cancers described hereinand stimulating anti-tumor immune activity. Thus, methods of treating acancer in a human subject are provided that comprise administering to asubject, any of the compositions as described above, in apharmaceutically acceptable formulation or carrier.

The subject being treated by the method is in one embodiment a subjectwho has a cancer that expresses FSHR, including those cancers listedabove. In another embodiment, the subject with FSHR-expressing cancer ortumor cells has been surgically treated to resect the tumor in questionprior to administration of the composition described herein. In oneembodiment, the subject is a female with ovarian cancer. In anotherembodiment, the female subject with ovarian cancer has been surgicallytreated to remove ovaries, fallopian tubes and/or uterus. Subjectshaving any of the other cancers enumerated above may be treated byappropriate surgery before or after application of these methods.

In one embodiment, the subject is administered a composition comprisinga nucleic acid construct as described above. In another embodiment, thesubject is administered a composition comprising a chimeric protein asdescribed above. In one specific embodiment, the method of treatingcancer in a human subject comprises administering to a subject in needthereof the bi-specific protein comprising a ligand that comprises anFSHβ sequence, which ligand binds to human FSHR, linked to a ligand thatbinds to NKG2D. In another embodiment, the composition is a viral vectorcarrying the nucleic acid construct to permit infection in vivo.

In another embodiment the method of treating cancer in a human subjectcomprises administering to a subject in need thereof a modified human Tcell that comprises a nucleic acid sequence that encodes a chimericprotein comprising a FSH sequence, a modification or fragment of saidFSH sequence, which ligand binds to FSHR, linked to nucleic acidsequences that encode T cell activating functions. In one embodiment,the T cell activating functions are provided by a nucleic acid sequencethat encodes an extracellular hinge domain, a transmembrane domain, aco-stimulatory signaling region, and a signaling endodomain. In oneembodiment, the modified T cell expresses any of the nucleic acidconstructs described herein. In one exemplary embodiment, the modified Tcell expresses the nucleic acid construct of FIG. 5 or similarconstructs described herein. In another embodiment, upon administrationthe modified T cell does not express Forkhead Box Protein (Foxp1). Inanother embodiment, the modified T cell carries a nucleic acid constructthat expresses or co-expresses sequences that ablate or decreaseexpression of Foxp1.

In still another embodiment, the modified human T cell is administeredwith clinically available PD-1 inhibitors. In still another embodiment,the modified human T cell is administered with clinically availableincluding TGF-β inhibitors (including blocking antibodies). In stillanother embodiment, the modified human T cell is administered withclinically available IL-10 inhibitors.

These methods of treatment are designed to enhance the therapeuticactivity of the T cells and prolong the survival of cancer patients. Thetherapeutic compositions administered by these methods, e.g., whethernucleic acid construct alone, in a virus vector or nanoparticle, aschimeric or bi-specific protein, or as modified anti-tumor T celltreated for adoptive therapy, are administered systemically or directlyinto the environment of the cancer cell or tumor microenvironment of thesubject. Conventional and pharmaceutically acceptable routes ofadministration include, but are not limited to, systemic routes, such asintraperitoneal, intravenous, intranasal, intravenous, intramuscular,intratracheal, subcutaneous, and other parenteral routes ofadministration or intratumoral or intranodal administration. Routes ofadministration may be combined, if desired. In some embodiments, theadministration is repeated periodically. In one embodiment, thecomposition is administered intraperitoneally. In one embodiment, thecomposition is administered intravenously. In another embodiment, thecomposition is administered intratumorally.

These therapeutic compositions may be administered to a patient,preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. The various components ofthe compositions are prepared for administration by being suspended ordissolved in a pharmaceutically or physiologically acceptable carriersuch as isotonic saline; isotonic salts solution or other formulationsthat will be apparent to those skilled in such administration. Theappropriate carrier will be evident to those skilled in the art and willdepend in large part upon the route of administration. Other aqueous andnon-aqueous isotonic sterile injection solutions and aqueous andnon-aqueous sterile suspensions known to be pharmaceutically acceptablecarriers and well known to those of skill in the art may be employed forthis purpose.

Dosages of these therapeutic compositions will depend primarily onfactors such as type of composition (i.e., T cells, vectors, nucleicacid constructs or proteins) the condition being treated, the age,weight and health of the patient, and may thus vary among patients. Inone embodiment, the modified T cell-containing composition isadministered in multiple dosages of between 2 million and 200 millionmodified T cells. Any value therebetween may be selected depending uponthe condition and response of the individual patient. As anotherexample, the number of adoptively transferred anti-tumor T cells can beoptimized by one of skill in the art. In one embodiment, such a dosagecan range from about 10⁵ to about 10¹¹ cells per kilogram of body weightof the subject. In another embodiment, the dosage of anti-tumor T cellsis about 1.5×10⁵ cells per kilogram of body weight. In anotherembodiment, the dosage of anti-tumor T cells is about 1.5×10⁶ cells perkilogram of body weight. In another embodiment, the dosage of anti-tumorT cells is about 1.5×10⁷ cells per kilogram of body weight. In anotherembodiment, the dosage of anti-tumor T cells is about 1.5×10⁸ cells perkilogram of body weight. In another embodiment, the dosage of anti-tumorT cells is about 1.5×10⁹ cells per kilogram of body weight. In anotherembodiment, the dosage of anti-tumor T cells is about 1.5×10¹⁰ cells perkilogram of body weight. In another embodiment, the dosage of anti-tumorT cells is about 1.5×10¹¹ cells per kilogram of body weight. Otherdosages within these specified amounts are also encompassed by thesemethods.

In another embodiment, a therapeutically effective adult human orveterinary dosage of a viral vector is generally in the range of fromabout 100 μL to about 100 mL of a carrier containing concentrations offrom about 1×10⁶ to about 1×10¹⁵ particles, about 1×10¹¹ to 1×10¹³particles, or about 1×10⁹ to 1×10¹² particles virus.

Administration of the protein-containing compositions may range betweena unit dosage of between 0.01 mg to 100 mg of protein (which isequivalent to about 12.5 μg/kg body weight).

Methods for determining the timing of frequency (boosters) ofadministration will include an assessment of tumor response to theadministration.

In still other embodiments, these methods of treating cancer byadministering a composition described herein are part of a combinationtherapy with various other treatments or therapies for the cancer.

In one embodiment, the methods include administration of a cytokine,such as IL-7 treatment as tumor-specific host conditioning strategies.Exogenous administration of IL-7 further promotes the in vivo activityspecifically of Foxp1-deficient T cells. In another embodiment, themethod further comprises administering to the subject along with thecompositions described herein, an adjunctive anti-cancer therapy whichmay include a monoclonal antibody, chemotherapy, radiation therapy, acytokine, or a combination thereof. In still another embodiment themethods herein may include co-administration or a course of therapy alsousing other small nucleic acid molecules or small chemical molecules orwith treatments or therapeutic agents for the management and treatmentof cancer. In one embodiment, a method of treatment of the inventioncomprises the use of one or more drug therapies under conditionssuitable for cancer treatment.

As previously mentioned surgical debulking, in certain embodiments is anecessary procedure for the removal of large tumor masses, and can beemployed before, during or after application of the methods andcompositions as described herein. Chemotherapy and radiation therapy, inother embodiments, bolster the effects of the methods described herein.Such combination approaches (surgery plus chemotherapy/radiation plusimmunotherapy) are anticipated to be successful in the treatment of manycancers along with the methods described herein.

In still further embodiments, the methods of treating a subject with anFSHR-expressing cancer or tumor include the following steps prior toadministration of the compositions described herein. In one embodiment,the methods include removing T cells from the subject and transducingthe T cells ex vivo with a vector expressing the chimeric protein. Inanother embodiment, the removed T cells are treated to ablate or reducethe expression of Fox-P1 in the T cells before or after transduction ofthe removed T cells with the nucleic acid construct described herein. Inanother method, the removed, treated T cells are cultured prior toadministration to remove Foxp1 from the cells ex vivo. Another methodstep involves formulating the T cells in a suitable pharmaceuticalcarrier prior to administration. It is also possible to freeze theremoved and treated T cells for later thawing and administration.

The methods of treatment may also include extracting T cells from thesubject for modification and ex vivo cell expansion followed by treatingthe subject with chemotherapy and depleting the subject of lymphocytesand optionally surgically resecting the tumor. These steps may takeplace prior to administering the modified T cells or other compositionsto the subject.

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only. Thecompositions, experimental protocols and methods disclosed and/orclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. The protocols and methods described inthe examples are not considered to be limitations on the scope of theclaimed invention. Rather this specification should be construed toencompass any and all variations that become evident as a result of theteaching provided herein. One of skill in the art will understand thatchanges or variations can be made in the disclosed embodiments of theexamples, and expected similar results can be obtained. For example, thesubstitutions of reagents that are chemically or physiologically relatedfor the reagents described herein are anticipated to produce the same orsimilar results. All such similar substitutes and modifications areapparent to those skilled in the art and fall within the scope of theinvention.

Example 1: Generation of Human and Mouse FSHR-Targeting Constructs

We generated a new fully murine construct as described herein againstmouse FSHR that includes the mouse version of all signals successfullyused in human patients. To target FSHR, we synthesized a constructexpressing a signal peptide, followed by the two subunits (alpha andbeta) of endogenous FSH, separated by a linker (See FIG. 1). Thistargeting motif was cloned in frame with a hinge domain from murine IgG,such as CD8α, followed by the transmembrane domain of CD8α, theintracellular domain of co-stimulatory mediator (e.g., murine 4-1BB orCD28), finally, the activating CD3ζ domain.

We have also generated constructs with the corresponding human sequences(see FIG. 5, Table 1, SEQ ID NOS: 1 and 2). Human variants of theFSHR-expressing constructs are generated to define the leadingformulation and demonstrate the relevance of experiments in mice. HumanHLA-A2+ T cells (>50% of Caucasians are A2+) from healthy donors aretransduced with retro- or lentiviral stocks containing the FSH-targetedconstruct, which is optimized for cytotoxic testing.

In frame constructs similar to the mouse sequences described above aregenerated to compare CD28 vs. 4-1BB/CD137. CD28 is an alternativeintracellular co-stimulatory motif because, although T cells expressing4-1BB/CD137 exhibited enhanced persistence in xenograft models inpublished experiments with CAR-T cells, it is unclear that long-termsurvival of T cells is preferable over multiple injections. In addition,the two human variants of the alpha subunit of human FSH (NM_000735.3vs. NM_001252383.1) are tested. These two subunits have differentlengths and have the potential to promote different binding affinities.

Overall, the 8 variants cloned (in frame) for expression into viralvectors are: 1) CGα (long)+4-1BB; 2) CGα (long)+CD28; 3) CGα(short)+4-1BB; and 4) CGα (short)+CD28 (see FIG. 8).

Other constructs are designed using only the beta subunit of FSH (whichprovides specificity for FSHR binding) and with a 15 aa binding regionof the beta subunit that also binds FSHR, e.g., the fragment of aminoacids 19-32 of SEQ ID NO: 2 of FSHβ 3 or other FSHβ fragments identifiedabove.

Example 2: FSH Constructs Respond Specifically to FSHR+ Tumor Cells

Retroviral (pSFG) vs. lentiviral (pELNS) vectors are tested to transducethe FSHR-carrying construct into human T cells. There is no formaldemonstration that lentiviral vectors are superior for ex vivotransduction. Most importantly, concerns regarding the risk ofinsertional oncogenesis after gene transfer in the T cell are negligibleafter a decade-long safety using retroviral vectors. The pSFG vector inparticular has been used many times for similar retroviral transductionof T cells in clinical trials^(41, 42).

Retroviral or lentiviral stocks expressing these constructs aregenerated and used to transduce human T cells from healthy HLA-A2 donors(>50% Caucasians). Retroviral stocks were used to transduceCD3/CD28-activated T cell splenocytes, which were FACS-sorted based onco-expression of GFP. The specificity of the binding of modified T cellsexpressing the FSH nucleic acid constructs of Example 1 was then testedagainst FSHR− or mock-transduced ID8-Defb29/Vegf-a ovarian cancer cells.As shown in FIG. 2, co-incubation of T cells transduced with the FHSRtargeting construct, but not T cells carrying an irrelevantmesothelin-targeting construct (K1), elicited the secretion of IFN-γ.Further supporting the specificity of FSHR recognition, IFN-γ secretiondid not occur in the presence of mock-transduced (naturally FSHR−) tumorcells.

Example 3: Intratumoral Administration of FSH Construct-Expressing TCells Delays the Progression of FSHR+ Breast Tumors

To gain some insight into the potential effectiveness and safety ofFSHR-targeting modified T cells in vivo in immunocompetent mice, we alsotransduced A7C11 breast cancer cells, a cell line generated from anautochthonous p53/KRas-mutated tumor, with mouse FSHR. Syngeneic micewere then challenged with flank tumors and administered identicallytreated 10⁶ FSHR-targeting modified T cells or mock transduced T cellsthrough intraperitoneal injection. As shown in FIG. 3, a singleadministration of FSHR-targeting modified T cells was sufficient tosignificantly delay the progression of established flank tumors, withoutnoticeable side effects. These results support the use of FSHR-targetingmodified T cells against ovarian orthotopic tumors, alone or incombination with other clinically available immunotherapies.

The use of mouse FSH as a targeting motif is much more predictive of theeffects of FSHR-targeting modified T cells than the use of human FSHRtargeting constructs in immunodeficient mice, because: 1) T cellsexpressing mouse FSH target potentially unidentified healthy cellsexpressing endogenous FSHR (unlike T cells expressing human FSHadministered into immunodeficient mice); 2) certain T cells, e.g.,CER-T, cells can boost polyclonal anti-tumor immunity by enhancingpre-existing T cell responses through antigen spreading and decreasingthe immunosuppressive burden; and 3) interactions between FSH and itsspecific receptor are highly conserved.

To demonstrate the cytotoxic potential of the FSHR-construct-containingT cells specifically against FSHR+ tumor cells, we again incubated theFSHR-constructs or mock-transduced T cells with FSHR+ID8-DeJb29/Vegf-a33(ovarian tumor) or A7C1134 (a cell line generated in the lab fromautochthonous p53/KRas-mutated breast tumors34) cells (40:1 ratio for 24h.), and cytotoxic killing was determined by counting Tripan blueneg(live) tumor cells. As shown in FIGS. 7A and 7B, FSH CER T cells, butnot mock-transduced lymphocytes eliminated both types of tumor cells.Comparable results were obtained in a MTS assay (not shown), furthersupporting that FSH targeting motifs are able to elicit CER-mediated Tcell cytotoxic activity in a FSHR-specific manner.

Example 4: The Effectiveness Vs. Toxicity of FSH Ligand-ExpressingModified T Cells in Preclinical Ovarian Cancer Models in ImmunocompetentHosts

To define the immunological consequences of using FSHR-targetingmodified T cells in immunocompetent preclinical tumors models thatinclude all healthy tissues where endogenous FSH could potentially bind(as it will happen in patients), we have generated new nucleic acidconstructs with the mouse counterparts of all targeting and activatingdomains. See Table 3, FIG. 6C and SEQ ID NOs 4 and 5.

We test the hypothesis that FSHR-targeting modified T cells showselective activity on the FSHR-expressing cells, impairing tumorprogression, while not harboring significant adverse effects in themouse. These results define the effect of this promising therapy onovarian cancer and ensure its safety for the future translation of thisapproach to the clinic.

We use aggressive orthotopic ID8-Vegf/DeJb29 tumors which have beentransduced and selected with mouse FSHR. We validate the generalapplicability of selected findings using transduced parental ID8 cellsand/or, an autochthonous p53-dependent inducible tumor model or ourderived cell lines.

Example 5. Mouse Clinical Trial: Define the Effectiveness ofFSHR-Targeting Modified T Cells Against Human Ovarian Cancers

We generated constructs with all human domains (see FIG. 5), which areused in our established cohort of primary tumor-derived xenografts inNSG mice. By using the same patients' tumors and T cells, we mimic alimited clinical trial that recapitulates the heterogeneity of clinicalovarian cancer. We hypothesize that FSHR-targeting modified T cells areeffective against established FSHR+ ovarian cancers from differentpatients, and synergize with combinatorial interventions targeting theTME.

By establishing the effectiveness and potential toxicity ofFSHR-targeting modified T cells in a variety of preclinical models, weprovide a mechanistic rationale for the testing of such T cells as apotential therapy against ovarian cancer. By using FSHR as a specifictumor target and a ligand comprising an FSH sequence as targeting motif,we overcome some of the challenges that have prevented the success of avariety of technologies against epithelial tumors.

To define full spectrum of activities of FSHR-targeting modified T cellsin vivo in immunocompetent hosts, we have generated fully murineFSHR-targeting modified T cells. (CD45.2+) established (FSHR+ and FSHR−)ovarian tumor-bearing mice (n≥10/group) are treated with (CD45.1+,congenic) 10⁶ i.p. anti-FSHR-targeting modified T cells 7 d. after tumorchallenge. Control mice receive mock transduced T cells. We comparesurvival, as an indisputable readout of effectiveness. Additionalinjections of FSHR-targeting modified T cells are administered dependingon these results. In different mice, at days 14, 21 and 28 after tumorchallenge, we track the homing and persistence of (specifically CD45.1+)transferred CD4 and CD8 T cells. Samples from spleen, draining(mediastinal) lymph nodes, bone marrow and tumor beds (peritoneal wash)are included. An exhaustive analysis of activation (e.g., CD44, CD69,CD27, CD25) vs. exhaustion_(e.g., PD-1, Lag3) markers in transferredlymphocytes is included. In addition, possible central memorydifferentiation of (congenic) FSHR-targeting modified T cells in BM andlymph nodes is analyzed, as a potential predictor of long-termengraftment and durable protection.

Example 6: Effects of FSHR-Targeting Modified T Cells on Pre-ExistingAnti-Tumor Immunity

We determine the effects that FSHR-targeting modified CER-T cells haveon ongoing anti-tumor immune responses, through antigen spreading andreduction of the immunosuppressive burden. For that purpose, we treattumor-bearing mice with FSHR-targeting modified CER-T cells vs.mock-transduced T cells and FACS-sort the endogenous (CD3+CD8+CD45.2+) Tcells from tumor and lymphatic locations, at days 7 and 14 afteradoptive transfer (days 14 and 21 after tumor). Magnitude of anti-tumorimmune responses_attributable to pre-existing T cells influenced bytreatment, are quantified through IFNγ and Granzyme B ELISPOT analysis.Activation vs. exhaustion markers in endogenous T cells additionallydefine the effects of FSHR-targeting modified T cells. To define immuneprotection against recurrences potentially elicited by thesecombinatorial interventions, mice are re-challenged if they rejectedtheir tumors.

Example 7—Short and Long-Term Toxicity Potentially Induced byFSHR-Targeting Modified T Cells

The main concern in T cell adoptive transfer protocols in the short termis the occurrence of a cytokine release syndrome, a systemicinflammatory response that induces non-infective fever and is associatedwith high levels of TNFα and IL-6.

We first monitor temperature and cytokine levels in treated vs. controlmice, besides obvious signs of disease (e.g., ruffled fur). If acytokine release syndrome was a frequent occurrence, we define whetherthe use of corticoids or IL6 depletion could make it a manageable event.The FSHR has not been reported to be expressed in normal tissue outsideof the ovary or the testes; therefore, we expect no long term sideeffects. However, we monitor any macroscopic alterations in treated micefor up to 4 months after treatment.

Example 8—Mouse Clinical Trial in Patient-Derived Xenografts

We engraft fresh advanced ovarian carcinoma that we receive (˜2-3specimens/month). Fresh specimens are delivered through a courier within2 h. after resection, and ˜1 mm3 chunks are engrafted into the ovarianbursa of NSG (severely immunodeficient) mice, through an optimizedsurgical procedure. Importantly, we receive peripheral blood from thesame patients, and buffy coats are immediately cryopreserved. In thelast 2 months, we have challenged ˜35 mice with 7 different specimens.Most tumors become palpable and visible in ˜45 days although theirprogress is slow. Approximately 2-3 new fresh specimens arrive everymonth. We generate xenografts for ≥10 primary tumors from differentpatients. Mice are used to define the effectiveness of FSHR-targetingmodified T cells against heterogeneous human ovarian tumors. For that,we CD3/CD28-expand and transduce the FHSR-targeting construct on T cellsfrom the same patient, thus mimicking its potential clinicalapplication.

We have generated FSH-targeting modified T cells with the correspondinghuman endogenous FSH, the hinge and transmembrane domains, and theco-stimulatory 4-1BB and activating CD3ζ motifs shown in FIG. 1 (allhuman sequences—see FIG. 5). We determine the expression of FSHR in eachxenograft through Western-blot analysis, to define how it predictseffectiveness. T cells from healthy donors with matching HLAs can betransduced and transferred.

To define the effectiveness of FSHR-targeting modified T cells againstovarian cancer, we use peripheral blood autologous to our engraftedhuman ovarian cancer specimens (≥3 mice/tumor; 10 different patients). Tcells are expanded and transduced with human FSHR-targeting nucleic acidconstructs or the empty vector (and/or irrelevant human CAR T19), andadoptively transferred into xenograft-bearing NSG mice challenged at thesame time. Tumor growth is monitored through palpation and ultrasound,and mice are sacrificed when tumors protrude through the abdomen, orearlier if the mice show signs of distress or advanced disease. Tumorgrowth, metastases and survival are quantified as a readout ofeffectiveness. How tumor growth is affected as a function of theexpression of FSHR is defined, using WB analysis in matching surgicalspecimens. We dissociate tumor specimens to determine the accumulationof transferred T cells in the presence vs. the absence of the target(NSG mice do not have endogenous lymphocytes). Persistence ofFSHR-targeting modified T cells at bone marrow and lymph node locationsis determined and correlated with the expression of the targeted hormonereceptor (FSHR) and effectiveness.

Example 9: Modulating Immunosuppression to Enhance the Effect ofFSHR-Targeting Modified T Cells Activity Against Ovarian Cancer

A potential challenge of adoptive T cell transfer interventions againstsolid tumors is the prospect that immunosuppressive networks in the TMEabrogate the protective activity of exogenous T cells. To modulate thetumor microenvironment in order to decrease its immunosuppressiveeffects, and increase survival, adjunctive methods are applied with theFSHR-targeting modified T cells. To elicit tumor rejection and sustainedprotection, the FSHR-targeting modified T cells are combined with theadministration of clinically available PD-1 inhibitors (or PD1 blockersalone in control groups). Effectiveness as a function of PD-L1expression in tumor cells is monitored. Alternatively, we block otherimmunosuppressive pathways in the ovarian cancer microenvironment inmice receiving FSHR-targeting modified T cells, including TGF-0 (forwhich blocking Abs have been recently developed) and IL-10.

In summary, the above examples demonstrate that we targeted a G-proteincoupled receptor (FSHR) that is expressed on the surface of ovariancancer cells in most tumors and has not been used in T cell basedinterventions. To enhance both specificity and receptor:ligandinteractions, we use the endogenous hormone as a targeting motif,thereby providing a rationale for the clinical testing of FSHR-targetingmodified T cells carrying the human counterparts of these motifs. We usethe endogenous ligand (a hormone), as opposed to anti-FSHR antibody orantibody fragments as a ligand to ensure effectiveness or specificity.We show that modified T cells directed against FSHR are safe and do notinduce obvious toxicity in vivo.

Most relevant for this application, the cytotoxic activity of modified Tcells targeting FSHR+ tumor cells boosts pre-existing lymphocyteresponses through antigen spreading, thus enhancing polyclonalanti-tumor immunity. In addition, we define whether combinatorialtargeting of suppressive networks operating in the ovarian cancermicroenvironment unleashes both FSHR-targeting modified T cells andtumor-infiltrating lymphocytes from tolerogenic pathways that coulddampen their protective activity.

Thus, this application is innovative at multiple conceptual andexperimental levels. We leverage a collection of freshly, orthotopicallyengrafted primary ovarian cancer xenografts to reflect the heterogeneityof the human disease in terms of FSHR expression and responsevariability. Specifically, we anticipate the above studies todemonstrate that ovarian cancer-bearing mice treated with FSHR-targetingmodified T cells show significantly increased survival (even tumorrejection in some cases), compared to controls receiving mock-transducedT cells. Correspondingly, we identify FSHR-targeting modified T cells invivo in treated mice for relatively long periods, as opposed to controlT cells. Because the bone marrow is a reservoir of memory T cells in oursystem that is where we see niches of persistent FSHR-targeting modifiedT cells. FSHR-targeting modified T cells are less sensitive tomechanisms of exhaustion at tumor beads. Together, these results areinterpreted as evidence for the therapeutic potential of FSHR-targetingmodified T cells, and pave the way for subsequent clinical testing.

The data is anticipated to demonstrate that FSHR-targeting modified Tcells induce a significant boost in the suboptimal but measurableanti-tumor activity of pre-existing T cells, as quantified by ELISPOTanalysis. The combined activity of FSHR-targeting modified T cells andendogenous lymphocytes correspondingly confers protection againstrecurrence in those mice rejecting established tumors. This underscoresthe potential of FSHR-targeting modified T cells to elicit polyclonalimmunological memory against tumor relapse, even if tumors lose targetedFSHR in the process.

The data is expected to show absence of significant adverse effects inthe long-term (e.g., autoimmunity) for ovarian cancer, as expression ofFSHR is restricted to the ovary (including all nucleated cells). Wecannot rule out that the acute administration of FSHR-targeting modifiedT cells will result in flu-like symptoms, but it is unlikely that theywill cause a cytokine-release syndrome. Together, these data furthersupport using FSHR-targeting modified T cells in ovarian cancerpatients.

The data is anticipated to demonstrate that >50% of primary tumorseventually grow exponentially in NSG mice and allow serial engraftmentinto different mice. 50-70% of these tumors express surface FSHR.Although we have shown the feasibility of engrafting human culturedovarian cells (which could be used as a back-up or complementaryapproach), this resource recapitulates the heterogeneity of the humandisease.

The data is anticipated to show that FSHR-targeting modified T cells arealso effective against xenografted human ovarian cancers that expressFSHR, but not against FSHR− tumors. Correspondingly, we anticipate thattumors with higher levels of FSHR expression are superior responders.Accordingly, we find enhanced persistence of modified T cells, strongerinfiltrates (due to FSHR-induced proliferation) and less exhaustion inFSHR^(high) tumor-bearing hosts. These results further support both thespecificity and the therapeutic potential of FSHR-targetingcompositions, as described herein.

Combination of PD-1 inhibitors with FSHR-targeting modified T cellspromote the rejection of PD-L1+ tumors, while only a significant delayin malignant progression is observed using individual treatments. Giventhe emerging clinical success of PD-1 blockers, we expect that they areoverall superior to other interventions targeting immunosuppression,which could nevertheless be more effective in PD-L1-tumors.Combinatorial interventions integrating cellular and molecularimmunotherapies has obvious implications for subsequent clinicaltesting.

Example 10—Expression of CER Variants in Human T Cells

We use peripheral blood from the aphaeresis of healthy HLA-A2+ donors(>50% of Caucasians), to minimize allogeneic reactions when transduced Tcells are co-incubated with (A2+) tumor cells. We use methods andresources as described^(5,29,30 44,45). Briefly, monocytes are depletedfrom the apheresis product and T cells are expanded in 5% normal humanAB serum using beads conjugated anti-CD3 (OKT3) and anti-CD28 (clone9.3) antibodies (3:1 bead/CD3+ cell ratio). On day 1 after stimulation,T cells are exposed to retro- or lentivirus supernatant encoding theFSH-targeted constructs variants (MOI˜3), on retronectin coated platesin the presence of 50 UI/mL of IL-2 and 1 ng/mL of IL-7, followed byspinoculation (1000 g, 45 min, 4° C.). On day 2, media are changed andspinoculation repeated, followed by up to 12 days of expansion. Aftercompletion of cell culture, magnetic beads are removed through magneticseparation and the cells are washed and resuspended in PlasmaLyte A(Baxter). The efficiency of transduction is determined by flow cytometryusing primary antibodies against human FSHβ (Clone 405326) expressedoutside infected cells, and PE-labeled anti-mouse IgG as secondaryantibodies. The transduction of human HLA-A2+ T cells is optimized forcytotoxic testing of all variants.

Example 11—the Anti-Tumor Activity of FSH-Targeted CER Variants In Vitro

An in vitro luciferase assay is used to test the effectiveness, i.e.,cytotoxic activity, of the FSH-targeted T cell variants generated asdescribed in the Examples above. We use human HLA-A2+ OVCAR3 ovariancancer cells¹⁹, which are also known to express high levels ofFSHR^(20,46). By using HLA-A2+ OVCAR3 T cells (expressing the mostcommon HLA type present in 50% of the Caucasians and 35% ofAfrican-Americans), we can use T cells from a wide variety of donorswithout eliciting an alloimmune response. We thereby restrict theelicitation of cytotoxic killing to specific recognition of the FSHRreceptor. We also measure IFN-γ production by transduced T cells inresponse to FSHR-expressing tumor cells. The two FSHR-expressing T cellvariants that show the best combined cytotoxic activity and IFN-γproduction are selected for subsequent in vivo testing.

Transfection with the retroviral vector pBABE-luc-puro makes the OVCAR3cells express firefly luciferase spontaneously, and allow selection by apuromycin resistance gene.

Human HLA-A2+ FSH-targeted construct-transduced T cells as describedherein are used as effectors. If more than 60% of the T cells aretransduced, the T cells are considered ready to use in the assays. Ifless than 60% of the T cells are transduced, we enrich for thetransduced T cells by FACS-sorting for the FSH+ cells.

After clonal selection and expansion of luciferase expressing OVCAR3(OVCAR3-luc), we plate 10,000 cells per well and coculture with T cellsexpressing the different FSH construct variants at ratios 1:1 (tumorcells:T cells), 1:5, 1:10 and 1:20. We also have a condition without Tcells as negative control (no cell death) and another treated withTriton X as positive control for maximal tumor cell death. 18 hoursafter plating the cells in co-culture we remove the media, wash thewells, lyse the cells and add the luciferase substrate. We measure theamount of luciferase signal to determine the specific lysis ofOVCAR3-luc cells by the FSH-targeted CER T cells.

Example 12—Production of IFN-Γ in Response to FSHR by all CER T CellVariants

Another parameter potentially important for therapeutic effectiveness isthe production of effector cytokines that may influence theimmunoenvironment in vivo.

Complementarily, we co-culture OVCAR3 cells and T cells expressing thedifferent variants of FSH-targeted construct and collect thesupernatants at 18 hours for determining IFNγ production by ELISA withat 1:1, 1:10 and 1:20 tumor cell to T cell ratios. If time permits, wealso determine IFN-γ production by intracellular staining ofFSH-targeted transfected T cells incubated with FSHR+ tumor cells, aswell as other cytokines such as TNF-α (ELISA) and granzyme B(intracellular staining).

These experiments are run in (at least) triplicates and repeated 3times, to achieve statistical significance (P<0.05) using theMann-Whitney's test. We identify two FSH-targeted construct-transfectedT cell variants for further investigation and development in an in vivosystem. We select the FSH-targeted CER variant that provides, in orderof priority: 1) The highest % of specific OVCAR3-luc cell lysis at thedifferent ratios; 2) the highest levels of IFN-γ secretion are selected.Where there are different best candidates at different ratios, we choosethe one with the highest specific lysis at a lowest ratio. If allvariants show similar activity, we prioritize the use of retroviralvectors, the inclusion of CD28 instead of 4-1BB, and the shorter CGαvariant. If there are no differences we choose the variant that showshigher transduction efficiency.

If FSHR-targeted killing of OVCAR3 cells is suboptimal, we use Caov-3ovarian cancer cells (ATCC #HTB-75), which also co-express FSHR andHLA-A2. In this case, this cell line is used for subsequent in vivotesting.

Example 13—FSHR Construct Variants Tested In Vivo in Ovarian CancerXenografts

We compare the in vivo efficacy of the two leading CER T cell variantsidentified in the Examples above. OVCAR3 cells (HLA-A2+19; FSHR+20) areengrafted into the ovarian bursa of NSG (severely immunodeficient)mice⁹. Orthotopic tumor-bearing mice are treated with HLA-A2+ CER Tcells as follows. Tumor chunks (˜1 mm³) are derived from TOV-21G ovarianclear cell carcinoma cells implanted into the flank of immunodeficientmice engrafted into the ovarian bursa of NSG mice in ˜1 month. Thechallenged ovary was taken by malignant growth and compared to the leftcontralateral ovary. The tumor (figure not shown) was particularlyaggressive and grew in ˜21 days. We have also challenged mice witheither tumor chunks (right ovary) or single cell suspensions from thesame freshly dissociated primary ovarian cancer specimens.

To define the effectiveness of FSH-targeted construct-transduced T cellsagainst FSHR+ ovarian cancer, we use the two CER T cell variants thatshow the best combined cytotoxic activity and IFN-γ production. If allvariants show similar activity, we prioritize the use of retroviralvectors, the inclusion of CD28 instead of 4-1BB, and the shorter CGαvariant. Mice showing established orthotopic ovarian tumors of similarsize by ultrasound and/or palpation (≥5 mice/group) receive T cellstransduced with the two variants of FSHR-targeting construct-transduced,or mock-transduced, T cells as an alternative control. If <60%transduction is achieved, positively transduced T cells are FACS-sortedand allowed to rest for 2 h before treatment. 10⁷ FSHR-targetconstruct-transduced T cells/injection and two injections 14 days apart(i.p. in PBS) are administered for these analyses. Tumor growth ismonitored through palpation and ultrasound, and mice are sacrificed whentumors protrude through the abdomen, or earlier if the show signs ofdistress or advanced disease. Tumor growth, metastases and survival arefirst quantified as an undisputable readout of effectiveness.

Example 14—the Persistence of the Two CER T Cell Variants In Vivo inTumor-Bearing Mice

An important parameter associated with long-term protection is thepersistence of central memory FSH-targeted construct-transduced T cellsthat can produce new waves of T cell effectors upon tumor recurrence. Todetermine what CER T cell variant persists for longer time in vivo, weidentically treat different OVCAR3-growing mice (≥5 mice/group) withFSH-targeted construct-transduced T cells, and monitor where they gatherand persist. First, the accumulation of transferred T cells at days 14and 28 after adoptive transfer are determined through FACS analysisusing dissociated tumor tissue, bone marrow (a reservoir of centralmemory T cells), lymph nodes and spleen samples. We use human CD3, asNSG mice do not have endogenous lymphocytes. Their memory attributes(CD62L+CD45RA-CD122+CD127+) at lymphatic and BM locations aredetermined. If mice reject tumors upon the administration ofFSH-targeted T cells, we re-challenge them with OVACR3 flank tumors, andcompare tumor progression with that in naïve (untreated) NSG mice.

Based on our previous observations, we anticipate that 5 mice per groupshould provide a 5% significance level and 95% power to detectdifferences of 20% or greater, using Mann-Whitney's or Wilcoxon's tests.Experiments use at least 5 mice/group (plus a repetition) and areanalyzed according to these statistical parameters. We thereby identifythe lead FSH-targeted T cell variant that is used for final preclinicaloptimization. The selected candidate shows a combination of, in order ofimportance: 1) strongest effectiveness against established tumor growth;2) central memory differentiation at lymphatic or bone marrow locations;and 3) superior overall persistence in vivo.

We theorize that persistence of FSH targeted T cells is important forlong-term remissions, based on clinical evidence treatingleukemia^(12,38,48). If both FSH target construct-transduced T cellvariants express CD28 and do not persist for at least 2 weeks, we alsotest the in vivo effectiveness of T cells carrying the constructs using4-1BB. If comparable tumor reduction was achieved, we would select CD28as a co-stimulatory domain.

We complement these experiments with the use of Caov-3 ovarian cancercells, which express even higher levels of the FSH Receptor and areHLA-A2⁺.

Example 15: Maximum Tolerated Dose (MTD) for Single Dose Administrations

The experimental plan to define MTD involves: (1) administering a singledose (2×10⁶, 10⁷ and 5×10⁷) mouse T cells in tumor-bearingimmunocompetent mice; (2) administering a single dose (2×10⁶, 10⁷ and5×10⁷) human T cells in human tumor-bearing immunodeficient mice; and(3) administering a single dose (2×10⁶, 10⁷ and 5×10⁷) mouse T cells intumor-free immunocompetent mice. After obtaining a single dose MTD fromthese experiments, the following experiments are conducted: (4)administering multi-doses of MTD (mouse T cells) in tumor-bearingimmunocompetent mice on days 21, 28 and 32; (5) administeringmulti-doses of MTD (human T cells) in human cancer-bearingimmunodeficient mice (3 times, a week apart); and (6) administeringmulti-doses of MTD (mouse T cells) in tumor-free immunocompetent mice ondays 0, 7 and 14. The results of these experiments define the multi-doseMTD.

These experiments provide a rationale for subsequent development ofFSH-targeted CER T cells for the treatment of ovarian cancer. They allowthe identification of a lead variant to be used for clinicalinterventions. To pave the way for immediate clinical testing, wedetermine the single-infusion maximum tolerated dose (MTD) of ourleading FSH-targeted CER. We evaluate tumor-dependent, FSHreceptor-specific, and non-specific toxicity after infusion ofFSH-targeted CER T cells.

Single Dose Escalation In Vivo in Immunocompetent Mice.

In order to determine single infusion MTD, a single dose escalation isconducted in both tumor- and non-tumor bearing immunocompetent mice.Thus, although the NOD/SCID/γc−/− (NSG) mouse is the best availablemodel for evaluating preclinical efficacy of CER T cells forFSHR-expressing tumors, the mouse tumor xenograft model is anon-relevant species for determination of some aspects of toxicology forthis particular CER T cell, because the human FSH component may not bindto normal mouse FSHR, and therefore, this mouse could under-predicttoxicity. For this reason, we first use FSH-targeted CER T cellconstructs that contain the exact mouse counterpart of each motifidentified, to be expressed by murine primary T cells. The goal of theseinitial experiments is to have a system where the endogenous (mouseFSHR) hormone receptor is present in the ovary and potentiallyunidentified healthy tissues. These studies test the capacity of thehost for orchestrating inflammatory reactions similar to what could beobserved in cancer patients in the presence of an intact immune system.Expression of FSH-targeted CER in mouse primary T cells is performedwith retroviral vectors independently of the results of Phase I, aslentiviruses do not infect mouse lymphocytes. Doses have been selectedbased on 5 years of experience with adoptive transfer of T cells invarious preclinical models^(7,30,43,49,50), including the use ofFSH-targeted CER lymphocytes to treat FSHR+ tumors in immunocompetentmice (FIG. 3). The “standard” efficacious and non-toxic dose is 10⁷cells, though we have shown efficacy below this dose. Mice are infusedwith 2×10⁶, 10⁷, or 5×10⁷ mock-transduced T cells, FSH-targeted CER Tcells, or HBSS. Because the expression of the FSHR has been alsoreported to be present in the altered endothelium found in metastaticlesions (but not in healthy blood vessels)⁵¹, as well as in theepithelial cells in prostate cancer⁵², we conduct two independentexperiments for each protocol; one used male mice and another usedfemale mice, with 5 mice per group in each experiment. These experimentsdefine toxicity in both genders by not excluding the potential presenceof endogenous FSHR in healthy male or female tissues.

In both males and females, FSHR-transduced ID8-Defb29/Vegf-a ovariancancer cells are administered to generate ovarian tumors disseminatedthroughout the peritoneal cavity. These tumors, albeit more slowly andslightly less reproducibly, also grow in male mice. Tumor-bearing miceare infused with T cells or HBSS 5 days after tumor injection. Becausethe standard administration of other CAR T cells in the clinic involvesprevious lymphodepletion²⁸, we sublethally irradiate the mice (300 rads)5 h before T cell adoptive transfer. All tumor cells and T cells areinitially infused i.p., because this is the route endorsed by the NCIfor targeting ovarian cancer in the clinic⁵³. If two or more mice at aparticular T cell dose show signs of toxicity, all mice at that dose andpaired HBSS treated controls were sacrificed for analysis. In that case,the administration of CER T cells i.v. is evaluated.

Single Dose Escalation In Vivo in Human Ovarian Cancer-Bearing Mice.

These complementary experiments are conducted in OVCAR3 (FSHR+) ovariancancer-bearing NSG mice, which is the best available model forevaluating preclinical efficacy of CER T cells for FSHR-expressingtumors (survival). FSH-targeted CERs are expressed with the viral vectordeveloped for clinical testing. Monocyte-depleted human T cells from theaphaeresis of HLA-A2+ healthy donors are used for transduction of the(human) FSH-targeted CER T cell variant. Because NSG mice do not have T,NK or B cells, adoptively transferred T cells undergo homeostaticexpansion anyway, making lymphodepletion (sublethal irradiation)unnecessary. The goal of these studies is to identify potential sideeffects restricted to the use of human T cells. For instance human IL-6is known to signal on mouse receptors and therefore a potential cytokinerelease syndrome could be detected.

In both sets of experiments, the following readouts are monitored:

The health status of mice is monitored and graded on a scale from 1 to4: 1—normal and healthy; 1.5—some lethargy, walking a bit slowly;2—moving slowly and a slight dragging of a limb; 2.5—dragging limbs whenmoving; 3—hunched posture and little movement; 3.5—laying on side, no orlittle movement upon touch; 4—death. A cohort of mice (≥5/group) issacrificed between 6 and 20 hours after T cell infusion and their healthstatus is recorded. Potential differences in the presence CER vs. HBSS,and between the presence vs. the absence of tumor are recorded. If miceexperienced signs of grade 3-4 health deterioration, serum is collectedfor quantification of IL-6 circulating levels, as this cytokine isresponsible for the cytokine release syndrome observed in some patients.

We also measure and record body weight on each day of T cell infusionand the three subsequent days. We continue to monitor body weightthroughout the experiments. If mice experienced >10% of body weight lossin 24 h they are sacrificed. Otherwise, recorded body weight is comparedto control mice, and also in the presence vs. the absence of a tumor.

Tissue sections are be analyzed in a blinded manner in all sacrificedmice. The liver, pancreas, spleen, small intestine, large intestine,heart, kidney, and lung are examined for evidence of tissue damage inH&E staining as well as the presence of (CD45+) inflammatory infiltratesby IHC. Leukocyte accumulation in healthy tissues is compared in micetreated with CER T cells vs. HBSS, and also in the presence vs. theabsence of tumor. The presence of red blood cells in the alveolar spaceor airways is additionally monitored to detect acute bleeding.

Survival is monitored in tumor-free vs. tumor-bearing mice receiving asingle infusion of CER vs. control T cells. If mice infused with thehighest dose (5×10⁷ cells) of FSH-targeted CER T cells suffer fromsevere acute toxicity, they are sacrificed regardless of whether theyhave tumors.

These experiments define the maximum tolerated dose for single infusionof our FSH-targeted T cells.

Maximum Tolerated Dose for Multi-Dose Administrations

The single dose MTD is likely between 2×10⁶ and 5×10⁷ of FSH-targetedCER T cells. To determine the toxicity of multiple FSH-targeted CER Tcell administrations analogous to a therapeutic regimen in a clinicalsetting tumor-dependent, FSH receptor-specific, and non-specifictoxicity are evaluated after infusion of FSH-targeted CER T cells. Bothhuman xenografts and immunocompetent mouse systems are used to predictpotential side effects in a clinical setting.

To determine whether multiple infusions at this dose would result intoxicity based on in vivo accumulation of T cells or host sensitization,tumor-bearing and non-tumor bearing mice are treated with threeinfusions of the MTD of CER T cells, the same amount of mock transduced(control) T cells, or HBSS. FSHR-transduced ID8-Defb29/Vegf-a ovariancancer-bearing immunocompetent mice receive primary mouse T cellstransduced with the mouse version of our FSH-targeted CER.Immunodeficient mice growing FSHR+HLA-A2+ OVCAR3 human ovarian cancercells are treated with human HLA-A2+ FSH-targeted CER T cells. Tomaximize the probability of tumor-associated toxicity, mice are treatedat later time points, once tumors have been established. Mice withorthotopic ID8-Defb29/Vegf-a ovarian tumors start treatment at day 21after tumor challenge, when ascites becomes evident in the absence oftreatment. Injections are repeated 7 days apart (days 28 and 32 aftertumor challenge). For OVCAR3 tumor-bearing mice, treatments areinitiated when tumors become palpable or are clearly established, asdetermined by ultrasound. Subsequent injections are administered a weekapart. NSG mice receiving human T cells are not expected to show signsof GVHD before 30 days of the first infusion, which provides anevolution time long-enough to define toxicity as a function ofeffectiveness. By treating tumors at advanced stages, we anticipate asignificant survival benefit for mice treated with FSH-targeted CER Tcells but we do not expect total tumor elimination. NSG mice treatedwith human CER T cells are sacrificed at day 28 after the first T celladministration. Again, 5 mice per group are used for each experiment.

The same readouts as described above, i.e., health status, body weight,histology and survival are monitored. We do not expect to identify signsof toxicity after the second and third administrations. We compare howthe presence vs. the absence of a tumor influences toxicity. In theunexpected event that significant toxicity is observed upon repeatedinjections, we escalate down the second and third infusion, startingwith 50% of the previously determined MTD, and administering the fulldose in the last injection. Subsequent reductions (50%) of the two lastinjections are tested if toxicity persists. Body weight is assessedstarting on the day of each T cell infusion and three subsequent days.We monitor body weight throughout the experiment. We anticipate a slightdecrease (˜1%) in body weight one day after these infusions, likelyreflecting the stress of handling and injection. However, we do notexpect major weight losses. If they occur, we escalate down the secondand third injections. We collect liver, pancreas, spleen, smallintestine, large intestine, heart, kidney, and lung from all sacrificedmice and generate histological sections for analysis of obvious tissuedamage and inflammatory infiltrates. Histological patterns aftertreatment with control vs. CER T cells, and administration in thepresence vs. the absence of tumor are compared. Survival is monitored intumor-free vs. tumor-bearing mice receiving multiple doses of CER vs.control T cells. We do not expect differences in survival of non-tumorbearing mice. However, we anticipate that advanced tumor-bearing micereceiving multiple doses of FSH-targeted CER T cells will exhibitsignificantly longer survival than those treated with control(mock-transduced) T cells. Survival in all groups is recorded.

Example 16—Evaluation of Biomarkers

The ability to monitor the PK/PD of the infused T cells is important forinterpretation of outcomes, determination of mechanism, andidentification of potential adverse effects early in the clinic. Inaddition, an important consideration is that the CER can becomeimmunogenic^(31,32). The expression of an endogenous hormone in ourFSH-targeted CER minimizes those potential side effects. Novel epitopesare created at the fusion joint of human signaling domains that are notnormally juxtaposed (e.g., the joint regions of CD28 and CD3ζ, or thejoint regions of CGα and the hinge region). Immunogenicity of the CERcan lead to the rejection of the adoptively-transferred T cells andcause inflammatory reactions. As a means to understand the in vivofunction of FSH-targeted CER T cells and those theoretical side effects,we evaluate specific biomarkers of cell activity in the serum. Serum isanalyzed using ELISA and standard assays for the following markers:

Cytokines are important predictors of both in vivo activity of CAR Tcells against tumor cells (e.g., tumor lysis) and potentialallergic/inflammatory reactions. The following inflammatory cytokinesare determined by ELISA in the serum of treated and control mice: IL-6,IFN-γ and TNF-α. From those, IL-6 is expected to show the strongestcorrelation with obvious behavioral alterations or changes in bodyweight, based on clinical evidence⁵⁴. We anticipate some increases in atleast IL-6 within the first 3 days after CER T cell administration,compared to the infusion of HBSS. This presumed mechanism of toxicity iswell-understood in the clinic, and effective interventions (usuallysteroidal or IL-6 blockers) are known and commonly practiced. Theseinflammation markers are useful in clinical trials to monitor patientsand determine the initial dose. We do not expect sustained elevations ofsystemic inflammatory cytokines >3 days after treatment, although theyare monitored nevertheless. Potential changes are recorded andcorrelated with clinical responses. These surrogate markers are used fordetermining the minimal anticipated biological effect level.

Hyperferritinemia, peaking at days 2-3, is another important surrogatemarker of a cytokine released syndrome in the clinic⁵⁷. We determine theconcentration of serum ferritin in control vs. treated mice, andcorrelate those levels with health deterioration, weight loss andhistological changes.

Creatinine is measured as an indicator of potential damage of renalfunction. Values in control mice are compared to those in mice receivingFSH-targeted CER T cells. Again, we do not anticipate any kidney damagedue to CER T cell administration.

AST is determined as an indicator of potential damage to the liver.Histological analyses of liver tissues are correlated with AST values,including potential tumor growth in the liver, as ovarian cancer is aperitoneal disease. Values in control mice receiving HBSS are comparedto those in mice infused with CER T cells. Based on clinical evidencewith other CER formulations, we do not expect that treatment withFSH-targeted T cells will adversely affect the liver.

We believe, based on clinical information that an important predictor oflong-term protection when targeted tumor determinants are truly specificis the persistence of adoptively transferred T cells. We analyze theaccumulation of FSH-targeted CER T cells in lymph nodes, the bonemarrow, and tumor tissue (if tumors are not rejected) of differentxenograft-bearing NSG mice (≥5/group). By analyzing dissociated lymphnodes and bone marrow at days 7 and 14 after adoptive transfer (beforeGVHD takes place56), flow cytometry determines the phenotype of (CD3+,as NSG mice do not have T or B cells) persistent CER T cells in terms ofacquisition of memory attributes (CD45RA-CD62L+CCR7+CD122+ lymphocytes).Thus, although immunodeficient mice are likely permissive, the presenceof a co-stimulatory domain in transferred human T cells promotes longterm engraftment and memory differentiation. This is interpreted as apredictor of subsequent therapeutic effectiveness but may have areflection in the levels of circulating cytokines.

For additional safety, mass doses much lower than the toxic dose areinitially used in mice, although the dose is slowly escalated aspatients are monitored for signs of toxicity. In one known study, themaximum tolerated dose (MTD) in human mesothelioma-bearing NSG micetreated with anti-mesothelin CAR T cells was 50×10⁷ cells/mouse. Weanticipate similar or better results, given the specificity of ourtarget. Based on preclinical evidence with different T cell adoptivetransfer protocols^(7,30,43) administration is up to 3 weeklyinjections. However, clinical protocols are based on infusing CAR Tcells in the course of 3 days²⁸. If significant toxicity is observedafter the third weekly injection, doses of CER T cells are adjusted fortolerance when administered for 3 consecutive days.

Example 17—Minimal Anticipated Biological Effect Level (MABEL)

The minimum anticipated biological effect level for humans is based onthe lowest animal dose or concentration required to produce activity invivo and/or in vitro data in animal/human systems. MABEL is definedthrough dose-response data from in vivo studies in human tumor-bearingNSG mice treated with CER T cells. Dose/concentration-effect curves aregenerated derived from experimental data and extrapolated from animal tohuman to initiate careful dose escalations. The starting point is thedose that corresponds to the minimal biological effect, using thebiomarkers defined above as surrogate markers.

To determine MABEL different NSG mice are challenged with OVCAR3 (FSHR+)ovarian cancer cells injected into the ovarian bursa (n≥5/group). Whentumors become palpable and show similar size by ultrasound (˜300 mm3),FSH-targeted CERs are expressed with the viral vector inCD3/CD28-expanded human T cells from the aphaeresis of HLA-A2+ healthydonors. A selected (human) FSH-targeted CER T cell variant is transducedand the maximum dose of CER T cells with no observed adverse effects isadministered. Control mice receive HBSS. IL-6, IFN-γ, TNF-α and ferritinare again determined in serum at the temporal points where increases areobserved (i.e., expected to happen only within the first 3 days). Basedon this baseline, different cohorts of tumor-bearing mice areidentically treated with doses of CER T cells escalated down by 50%,until any increases in the aforementioned cytokines (compared to controlmice) disappears (becomes the same as in the control group). This amountof FSH-targeted CER T cells, calculated in terms of body weight, is usedto define the starting dose for human intervention.

Patients currently receive between 10⁷ and 10⁸ T cells transduced withdifferent CER per kg of body weight in ongoing trials²⁸. Consideringthat we have not observed noticeable toxicity in mice at doses of 10⁹/kgof body weight (˜10⁷ CER T cells/mouse; ˜30 g/mouse; FIG. 3), weanticipate that a safe initial dose can be adjusted and escalated indifferent patients to reach a “No Observable Adverse Effect Level” belowthese amounts. For additional safety, a “split dose” approach to dosingis followed over 3 days, administering CER-transduced T cells using 10%of the total intended on day 0, 30% on day 1 and 60% on day 2, starting2 days following chemotherapy²⁸.

We analyze test batches of the initial virus to determine which clone isproducing a high titer of FSH-targeted CER virus, by both qPCR analysisand ELISA for human FSH. We transduce human T cells with this virus, anddetermine the expression of FSHβ, CD3, CD4 and CD8 by flow cytometry,and IFN-γ production after co-culture with FSHR+ OVCAR3 tumor cells (asin FIG. 2). We select the cell clones that produce the highest titersfor expansion, testing, and production of the master cell bank. Thismaster cell bank can be used as a source of virus producing cells foradditional studies.

We then test the product to assure the safety of biological productsincluding tests for (1) sterility, (2) mycoplasma, and (3) adventitiousviral agents, following FDA guidelines.

Example 18—Persistence of CER-T Cells in Peripheral Blood

A Q-PCR assay for determining the trafficking and persistence ofadoptively transferred CER T cells in vivo in peripheral blood isdefined. Primers and TaqMan probes are designed to span the jointsbetween the sequence of CGα and the transmembrane domains of the CER,which are not naturally present in any cell in patients. In addition, aflow cytometry analysis of transferred CER T cells is optimized based onthe detection of FSHβ on transduced (CD3+) T cells, by fluorescentlylabeling available anti-human FSH antibodies, or using a primaryanti-FSH antibody and a fluorescently labeled secondary antibody.Tracking of transferred T cells includes CD4 and CD8 antibodies in theassay, to gain understanding of the mechanisms of therapeuticeffectiveness. These reagents are tested in a new cohort of OVCAR3tumor-bearing NSG mice (≥5) adoptively transferred with A2+ T cells fromhealthy donors transduced with the clinical grade vector and procedures.This experiment verifies the effectiveness of the new reagent againsttumor growth.

The primary toxicity anticipated is whether the T cells will causeinflammation by killing tumor cells that express the target. Aftersetting the starting dose, the conventional dose escalation isconservatively based on 3-fold increments. Because we use endogenousFSH, the risk of anaphylaxis or immune targeting of CAR T cellspreviously described for xenogeneic (murine) scFvs is negligible. Asimportantly, the expression of the FSH receptor has been limited to theovary through millions of years of evolution. Because they are routinelyresected in ovarian cancer patients and no other organ should bind theFSH hormone, we expect that FSH-targeted CER T cells represent a safeand effective intervention.

Example 19—Use of Tall Cells as a Universal Platform

CER constructs have been expressed in TALL-103/2 cells and in TALL-104cells (ATCC CRL11386; U.S. Pat. No. 5,702,702), to re-direct theircytotoxic potential towards FSHR+ tumors through ligand rather than ascFv, but otherwise using the activating domains successfully usedagainst leukemias.

The optimization of TALL-103/2 cells as an universal allogeneic platformis one embodiment, because they grow significantly faster than TALL-104cells in vitro and therefore will be easier to handle for massproduction. We will nevertheless also use TALL-104 cells, which trafficspontaneously to tumor beds. Expressing FSH-targeted chimeric receptorsenhances the therapeutic potential of TALL-103/2 and TALL-104 cells, byre-directing their cytotoxic activity towards FSHR+ ovarian cancer cellsthrough its endogenous (non-immunogenic) ligand. Preliminary resultsshow that transduction of our FSH CER empowers TALL-103/2 or TALL-104cells to kill ovarian cancer cells spontaneously expressing FSHRssignificantly more effectively than their mock-transduced counterparts.FSH-targeting CER TALL-cells are able to specifically and effectivelykill FSHR expressing ovarian cancer cells and abrogate malignantprogression in clinically relevant ovarian cancer models withoutsignificant adverse effects. These results are anticipated to translateto ovarian cancer patients in subsequent clinical trials.

We have already transduced and selected TALL-103/2 and TALL-104 cellswith pBMN retroviruses encoding human FSHR targeted CERs. To demonstratethat spontaneous (NK-like) cytolytic activity of TALL-103/2 cells can besignificantly enhanced through the expression of our FSH-targeted CER,we again performed in vitro cytotoxicity experiments using human ovariancancer (A2⁺FSHR⁺) OVCAR3 cells as targets. As shown in FIG. 12,mock-transduced TALL-103/2 cells showed, as expected, somedose-dependent anti-tumor activity against ovarian cancer cells.However, the expression of our FSH-targeted CER empowered this cell lineto eliminate ˜60% of tumor cells at effector:target ratios as low as1:4. These experiments support the potential of re-directing ouruniversal allogeneic platform more specifically against FSHR⁺ (70%)ovarian tumors through the expression of FSHR CERs. Of note,FSH-targeted CER TALL-103/2 cells grow in our bioreactors as effectivelyas parental cells. This experiment provided proof-of-concept for apotentially safer and universally accessible system; namely, combiningthe spontaneous therapeutic activity of TALL-103/2 or TALL 104 cellswith the power of our FSH-targeted activating receptor, to maximizetheir specificity; and their anti-tumor cytotoxic activity.

Importantly, the spontaneous cytotoxic activity of TALL-103/2 cells inthe absence of CAR/CER expression is restricted to NK-susceptibletargets that express NKG2D ligands, such as K562 and U937 leukemiccells, while healthy cells are completely spared from cytotoxic killing.In addition, TALL-103/2 cells are unlikely to cause GVHD uponadministration into patients, because they express a single (γδ) TCR, asit was demonstrated for TALL-104 cells. However, the effector activityof TALL-103/2 cells can be elicited through the activation of CD3 or theadministration of IL-2. Together with their faster ex vivo growth inbio-reactors, compared to clinically available TALL-104 cells, theseattributes make TALL-103/2 cells potentially superior allogenicplatforms for re-directing their anti-tumor potential through theexpression of our FSH-targeted CERs. However, TALL-104 cells are alsodesirable for this use as they traffic spontaneously to tumor beds.

Each and every patent, patent application including U.S. ProvisionalPatent Application 62/059,068, U.S. Provisional Patent Application No.62/202,824, and any document identified herein and the sequence of anypublically available nucleic acid and/or peptide sequence citedthroughout the disclosure is expressly incorporated herein by referencein its entirety. Embodiments and variations of this invention other thanthose specifically disclosed above may be devised by others skilled inthe art without departing from the true spirit and scope of theinvention. The appended claims include such embodiments and equivalentvariations.

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The invention claimed is:
 1. A chimeric protein comprising i) ligandthat comprises an FSHβ subunit or a fragment thereof, which ligand iscapable of binding to a human follicle stimulating hormone (FSH)receptor on a tumor that expresses FSH receptor, ii) an extracellularhinge domain, iii) a transmembrane domain, iv) a co-stimulatorysignaling domain, and v) a signaling endodomain.
 2. The chimeric proteinof claim 1, wherein the chimeric protein is capable of activating amodified human T cell expressing the chimeric protein.
 3. The chimericprotein of claim 1, wherein the ligand comprises a full length FSHβsubunit.
 4. The chimeric protein of claim 1, wherein the ligand furthercomprises an FSHα subunit.
 5. The chimeric protein of claim 4, whereinthe FSHβ subunit is linked to the FSHα subunit by a linker.
 6. Thechimeric protein of claim 1, wherein the ligand comprises an amino acidsequence of amino acids 19-129 of SEQ ID NO:
 2. 7. The chimeric proteinof claim 1, wherein the FSHβ subunit is a first FSHβ subunit, whereinthe ligand further comprises a second FSHβ subunit and a linker, andwherein the first FSHβ subunit is linked to the second FSHβ subunit bythe linker.
 8. The chimeric protein of claim 1, wherein the ligandcomprises an FSHβ subunit fragment.
 9. The chimeric protein of claim 8,wherein the FSHβ subunit fragment comprises an amino acid sequenceselected from the group consisting of amino acids 19-33 of SEQ ID NO: 2,51-71 of SEQ ID NO: 2, 69-83 of SEQ ID NO: 2, and 99-113 of SEQ ID NO:2.
 10. The chimeric protein of claim 1, wherein the extracellular hingedomain is selected from a CD8 hinge domain, a IgG1 hinge domain, a CD3hinge domain and a CH₂CH₃ region of an immunoglobulin.
 11. The chimericprotein of claim 1, wherein the transmembrane domain is selected from aT cell receptor, CD28, CD3 ε, CD45, CD4, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, and CD154 transmembrane domain.12. The chimeric protein of claim 1, wherein the co-stimulatory domainis selected from a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3 costimulatory domain. 13.The chimeric protein of claim 1, wherein the signaling endodomain isselected from a CD3ζ, TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5,CD22, CD79a, CD79b, and CD66d signaling endodomain.
 14. The chimericprotein of claim 1, further comprising a spacer element linking theextracellular hinge domain to the transmembrane domain.
 15. The chimericprotein of claim 1, wherein the extracellular hinge domain comprises aCD8 hinge domain, the transmembrane domain comprises a CD8 transmembranedomain, the co-stimulatory signaling domain comprises a 4-1BBcostimulatory domain, and the signaling endodomain comprises a CD3signaling endodomain.
 16. The chimeric protein of claim 1, wherein theligand comprises the FSHβ subunit linked to a FSHα subunit by a linker,the extracellular hinge domain comprises a CD8 hinge domain, thetransmembrane domain comprises a CD8 transmembrane domain, theco-stimulatory signaling domain comprises a 4-1BB costimulatory domain,and the signaling endodomain comprises a CD3 ζ signaling endodomain. 17.The chimeric protein of claim 1, wherein the ligand comprises the FSHβsubunit linked to a FSHα subunit by a linker, the extracellular hingedomain comprises a CD8 hinge domain, the transmembrane domain comprisesa CD8 transmembrane domain, the co-stimulatory signaling domaincomprises a CD28 costimulatory domain, and the signaling endodomaincomprises a CD3 ζ signaling endodomain.
 18. The chimeric protein ofclaim 1, wherein the tumor is ovarian cancer, prostate cancer, breastcancer, colon cancer, esophageal cancer, cervical cancer, pancreaticcancer, bladder cancer, kidney cancer, lung cancer, liver cancer,stomach cancer, or testicular cancer.
 19. The chimeric protein of claim2, wherein the modified human T cell is capable of binding to cells ofblood vessels of either primary or metastatic tumors.
 20. A chimericprotein comprising amino acids 19-129, 130-144, 313-336, and 337-378 ofSEQ ID NO: 2.